The plant uncoupling protein homologues: a new family of energy-dissipating proteins in plant mitochondria.

Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. As in mammalian tissues, UCPs are rather ubiquitous in the plant kingdom and widespread in plant tissues in which they could have various physiological roles, such as heat production or protection against free oxygen radicals. The simultaneous occurrence in plant mitochondria of two putative energy-dissipating systems, namely UCP which dissipates the proton motive force, and alternative oxidase (AOX) which dissipates the redox potential, raises the question of their functional interactions.     

Structural basis of the ribosomal machinery for peptide bond formation,translocation, and nascent chain progression

Crystal structures of tRNA mimics complexed with the large ribosomal subunit of Deinococcus radiodurans indicate that remote interactions determine the precise orientation of tRNA in the peptidyl-transferase center (PTC). The PTC tolerates various orientations of puromycin derivatives and its flexibility allows the conformational rearrangements required for peptide-bond formation. Sparsomycin binds to A2602 and alters the PTC conformation. H69, the intersubunit-bridge connecting the PTC and decoding site, may also participate in tRNA placement and translocation. A spiral rotation of the 3' end of the A-site tRNA around a 2-fold axis of symmetry identified within the PTC suggests a unified ribosomal machinery for peptide-bond formation, A-to-P-site translocation, and entrance of nascent proteins into the exit tunnel. Similar 2-fold related regions, detected in all known structures of large ribosomal subunits, indicate the universality of this mechanism.     

Yeasts associated with Manteca

Manteca is a traditional milk product of southern Italy produced from whey deriving from Caciocavallo Podolico cheese-making. This study was undertaken to obtain more information about the microbiological properties of this product and particularly about the presence, metabolic activities, and technological significance of the different yeast species naturally occurring in Manteca. High numbers of yeasts were counted after 7 days ripening (10(4)-10(5) cfu g(-1)) and then decreased to 10(2) at the end. A total of 179 isolates were identified and studied for their phenotypic and genotypic characteristics. The most frequently encountered species were Trichosporon asahii (45), Candida parapsilosis (33), Rhodotorula mucilaginosa (32), Candida inconspicua (29). Some of these yeasts showed lipolytic activity (32 strains) and proteolytic activity (29 strains), NaCl resistance up to 10% and growth up to 45 degrees C (42 strains). Biogenic amines were formed by proteolytic strains, in particular phenylethylamine, putrescine and spermidine. Spermidine was produced by all the yeasts tested in this work, but only Trichosporon produced a great quantity of this compound. Histamine was not detectable. Caseinolytic activity was common to almost all strains, corresponding to the ability to efficiently split off amino-terminal amino acids. The highest and most constant activity expressed by all species was X-prolyl-dipeptidyl aminopeptidase. The findings suggest that the presence of yeasts may play a significant role in justifying interactions with lactic acid bacteria, and consequently with their metabolic activity in the definition of the peculiar characteristics of Manteca cheese.     

The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria

Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H₂O₂ release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.     

Localization of angiotensin-II type 1(AT1) receptors on buffalo spermatozoa: AT1 receptor activation during capacitation triggers rise in cyclic AMP and calcium

The purpose of this study was to determine the role of Ang-II in buffalo spermatozoa; localize angiotensin type 1 (AT1) receptors on the sperm surface and understand the signaling mechanisms involved therein. Immunoblotting and immunocytochemistry using polyclonal Rabbit anti-AT1 (N-10) IgG were performed to confirm the presence of AT1 receptors. Intracellular levels of cyclic adenosine monophosphate (cAMP) were determined by non-radioactive enzyme immunoassay, while that of Calcium [Ca²⁺] were estimated by fluorimetry using Fura2AM dye. The results obtained showed that AT1 receptors were found on the post-acrosomal region, neck and tail regions. Immunoblotting revealed a single protein band with molecular weight of 40 kDa. Ang-II treated cells produced significantly higher level of cAMP compared to untreated cells (22.66 ± 2.4 vs. 10.8 ± 0.98 pmol/10⁸ cells, p < 0.01). The mean levels of Ca²⁺ were also higher in Ang-II treated cells compared to control (117.4 ± 6.1 vs. 61.15 ± 4.2 nmol/10⁸ cells; p < 0.01). The stimulatory effect of Ang-II in both the cases was significantly inhibited in the presence of Losartan (AT1 antagonist; p < 0.05) indicating the involvement of AT1 receptors. Further, presence of neomycin (protein kinase C inhibitor) inhibited significantly the Ang-II mediated rise in Ca²⁺ indicating the involvement of PKC pathway. These findings confirm the presence of AT1 receptors in buffalo spermatozoa and that Ang-II mediates its actions via the activation of these receptors. Ang-II stimulates the rise in intracellular levels of cAMP and Ca²⁺ during capacitation.     

In situ hybridization analysis of the temporospatial expression of the midkine/pleiotrophin family in rat embryonic pituitary gland

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke’s pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.     

The association between ownership of common household devices and obesity and diabetes in high,middle and low income countries

Background:

Household devices (e.g., television, car, computer) are common in high income countries, and their use has been linked to obesity and type 2 diabetes mellitus. We hypothesized that device ownership is associated with obesity and diabetes and that these effects are explained through reduced physical activity, increased sitting time and increased energy intake.

Methods:

We performed a cross-sectional analysis using data from the Prospective Urban Rural Epidemiology study involving 153 996 adults from high, upper-middle, lower-middle and low income countries. We used multilevel regression models to account for clustering at the community and country levels.

Results:

Ownership of a household device increased from low to high income countries (4% to 83% for all 3 devices) and was associated with decreased physical activity and increased sitting, dietary energy intake, body mass index and waist circumference. There was an increased odds of obesity and diabetes with the ownership of any 1 household device compared to no device ownership (obesity: odds ratio [OR] 1.43, 95% confidence interval [CI] 1.32–1.55; diabetes: OR 1.38, 95% CI 1.28–1.50). Ownership of a second device increased the odds further but ownership of a third device did not. Subsequent adjustment for lifestyle factors modestly attenuated these associations. Of the 3 devices, ownership of a television had the strongest association with obesity (OR 1.39, 95% CI 1.29–1.49) and diabetes (OR 1.33, 95% CI 1.23–1.44). When stratified by country income level, the odds of obesity and diabetes when owning all 3 devices was greatest in low income countries (obesity: OR 3.15, 95% CI 2.33–4.25; diabetes: OR 1.97, 95% CI 1.53–2.53) and decreased through country income levels such that we did not detect an association in high income countries.

Interpretation:

The ownership of household devices increased the likelihood of obesity and diabetes, and this was mediated in part by effects on physical activity, sitting time and dietary energy intake. With increasing ownership of household devices in developing countries, societal interventions are needed to mitigate their effects on poor health.The increasing global prevalence of obesity and type 2 diabetes mellitus has been driven predominantly by increases in high income countries.^1,2 However, increases are expected in low and middle income countries, due in part to rapid development and industrialization.Proximal determinants of obesity and diabetes include energy expenditure and intake;^3–5 however, the upstream factors are complex and entail numerous environmental factors. Of these, the increased use of common household devices (e.g., televisions, cars, computers) has been linked to increased sitting, decreased physical activity, obesity, metabolic syndrome and diabetes.^6–12 Time spent watching television has also been linked to poor diet¹³ and increased caloric intake.¹⁴ However, these findings are based on studies in high income countries where the ownership of these devices is common.^15,16 In low and middle income countries, such household devices are less prevalent, but their prevalence is rapidly increasing. Studies in countries with greater variability in the ownership of household devices are needed to understand the full effect of owning such devices on the risk of obesity and diabetes.We hypothesized that the ownership of a television, car or computer would be associated with an increased risk of obesity and diabetes and that these effects would be explained by reduced physical activity, increased sitting time and increased energy intake.     

Sphingolipid signalling mediates mitochondrial dysfunctions and reduced chronological lifespan in the yeast model of Niemann‐Pick type C1

The Niemann‐Pick type C is a rare metabolic disease with a severe neurodegenerative phenotype characterized by an accumulation of high amounts of lipids (cholesterol and sphingolipids) in the late endosomal/lysosomal network. It is caused by loss‐of‐function point mutations in either NPC1 or NPC2, which seem to mediate proper intracellular lipid transport through endocytic pathway. In this study, we show that yeast cells lacking Ncr1p, an orthologue of mammalian NPC1, exhibited a higher sensitivity to hydrogen peroxide and a shortened chronological lifespan. These phenotypes were associated with increased levels of oxidative stress markers, decreased levels of antioxidant defences and mitochondrial dysfunctions. Moreover, we report that Ncr1p‐deficient cells displayed high levels of long chain bases (LCB), and that Sch9p‐phospho‐T570 and Sch9p levels increased in ncr1Δ cells through a mechanism regulated by Pkh1p, a LCB‐activated protein kinase. Notably, deletion of PKH1 or SCH9 suppressed ncr1Δ phenotypes but downregulation of de novo sphingolipid biosynthesis had no protective effect, suggesting that LCBs accumulation may result from an increased turnover of complex sphingolipids. These results suggest that sphingolipid signalling through Pkh1p‐Sch9p mediate mitochondrial dysfunction, oxidative stress sensitivity and shortened chronological lifespan in the yeast model of Niemann‐Pick type C disease.     

Aberrant gene expression in the Arabidopsis SULTR1;2 mutants suggests a possible regulatory role for this sulfate transporter in response to sulfur nutrient status

Sulfur is required for the biosynthesis of cysteine, methionine and numerous other metabolites, and thus is critical for cellular metabolism and various growth and developmental processes. Plants are able to sense their physiological state with respect to sulfur availability, but the sensor remains to be identified. Here we report the isolation and characterization of two novel allelic mutants of Arabidopsis thaliana, sel1‐15 and sel1‐16, which show increased expression of a sulfur deficiency‐activated gene β‐glucosidase 28 (BGLU28). The mutants, which represent two different missense alleles of SULTR1;2, which encodes a high‐affinity sulfate transporter, are defective in sulfate transport and as a result have a lower cellular sulfate level. However, when treated with a very high dose of sulfate, sel1‐15 and sel1‐16 accumulated similar amounts of internal sulfate and its metabolite glutathione (GSH) to wild‐type, but showed higher expression of BGLU28 and other sulfur deficiency‐activated genes than wild‐type. Reduced sensitivity to inhibition of gene expression was also observed in the sel1 mutants when fed with the sulfate metabolites Cys and GSH. In addition, a SULTR1;2 knockout allele also exhibits reduced inhibition in response to sulfate, Cys and GSH, consistent with the phenotype of sel1‐15 and sel1‐16. Taken together, the genetic evidence suggests that, in addition to its known function as a high‐affinity sulfate transporter, SULTR1;2 may have a regulatory role in response to sulfur nutrient status. The possibility that SULTR1;2 may function as a sensor of sulfur status or a component of a sulfur sensory mechanism is discussed.