Increasing mitochondrial substrate-level phosphorylation can rescue respiratory growth of an ATP synthase-deficient yeast

In a previous study we have identified Fmc1p, a mitochondrial protein involved in the assembly/stability of the yeast F0F1-ATP synthase at elevated temperatures. The deltafmc1 mutant was shown to exhibit a severe phenotype of very slow growth on respiratory substrates at 37 degrees C. We have isolated ODC1 as a multicopy suppressor of the fmc1 deletion restoring a good respiratory growth. Odc1p expression level was estimated to be at least 10 times higher in mitochondria isolated from the deltafmc1/ODC1 transformant as compared with wild type mitochondria. Interestingly, ODC1 encodes an oxodicarboxylate carrier, which transports alpha-ketoglutarate and alpha-ketoadipate or any other transported tricarboxylic acid cycle intermediate in a counter-exchange through the inner mitochondrial membrane. We show that the suppression of the respiratory-growth-deficient fmc1 by the overexpressed Odc1p was not due to a restored stable ATP synthase. Instead, the rescuing mechanism involves an increase in the flux of tricarboxylic acid cycle intermediate from the cytosol into the mitochondria, leading to an increase in the alpha-ketoglutarate oxidative decarboxylation, resulting in an increase in mitochondrial substrate-level-dependent ATP synthesis. This mechanism of metabolic bypass of a defective ATP synthase unravels the physiological importance of intramitochondrial substrate-level phosphorylations. This unexpected result might be of interest for the development of therapeutic solutions in pathologies associated with defects in the oxidative phosphorylation system.     

Polymorphisms in human organic anion-transporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry

Organic anion-transporting polypeptide 1A2 (OATP1A2) is a drug uptake transporter known for broad substrate specificity, including many drugs in clinical use. Therefore, genetic variation in SLCO1A2 may have important implications to the disposition and tissue penetration of substrate drugs. In the present study, we demonstrate OATP1A2 protein expression in human brain capillary and renal distal nephron using immunohistochemistry. We also determined the extent of single nucleotide polymorphisms in SLCO1A2 upon analyses of ethnically defined genomic DNA samples (n = 95 each for African-, Chinese-, European-, and Hispanic-Americans). We identified six nonsynonymous polymorphisms within the coding region of SLCO1A2 (T38C (I13T), A516C (E172D), G559A (A187T), A382T (N128Y), A404T (N135I), and C2003G (T668S)), the allelic frequencies of which appeared to be ethnicity-dependent. In vitro functional assessment revealed that the A516C and A404T variants had markedly reduced capacity for mediating the cellular uptake of OATP1A2 substrates, estrone 3-sulfate and two delta-opioid receptor agonists, deltorphin II, and [D-penicillamine(2,5)]-enkephalin. On the other hand, the G559A and C2003G variants appeared to have substrate-dependent changes in transport activity. Cell surface biotinylation and immunofluorescence confocal microscopy suggested that altered plasma membrane expression of the transporter may contribute to reduced transport activity associated with the A516C, A404T, and C2003G variants. The A404T (N135I) variant also showed a shift in the apparent molecular size, indicative of alterations in glycosylation status. Taken together, these data suggest that SLCO1A2 polymorphisms may be an important yet unrecognized contributor to inter-individual variability in drug disposition and central nervous system entry of substrate drugs.     

Saturated fat-induced changes in Sf 60-400 particle composition reduces uptake of LDL by HepG2 cells

The ability of human postprandial triacylglycerol-rich lipoproteins (TRLs), isolated after meals enriched in saturated fatty acids (SFAs), n-6 PUFAs, and MUFAs, to inhibit the uptake of 125I-labeled LDL by the LDL receptor was investigated in HepG2 cells. Addition of TRLs resulted in a dose-dependent inhibition of heparin-releasable binding, cell-associated radioactivity, and degradation products of 125I-labeled LDL (P < 0.001). SFA-rich Svedberg flotation rate (Sf) 60-400 resulted in significantly greater inhibition of cell-associated radioactivity than PUFA-rich particles (P = 0.016) and total uptake of 125I-labeled LDL compared with PUFA- and MUFA-rich particles (P < 0.02). Normalization of the apolipoprotein (apo)E but not apoC-III content of the TRLs removed the effect of meal fatty acid composition, and addition of an anti-apoE antibody reversed the inhibitory effect of TRLs on the total uptake of 125I-labeled LDL. Real time RT-PCR showed that the SFA-rich Sf 60-400 increased the expression of genes involved in hepatic lipid synthesis (P < 0.05) and decreased the expression of the LDL receptor-related protein 1 compared with MUFAs (P = 0.008). In conclusion, these findings suggest an alternative or additional mechanism whereby acute fat ingestion can influence LDL clearance via competitive apoE-dependent effects of TRL on the LDL receptor.     

Attenuated protein expression vectors for use in siRNA rescue experiments

Transient transfection of small interfering RNA (siRNA) provides a powerful approach for studying cellular protein functions, particularly when the target protein can be re-expressed from an exogenous siRNA-resistant construct in order to rescue the knockdown phenotype, confirm siRNA target specificity, and support mutational analyses. Rescue experiments often fail, however, when siRNA-resistant constructs are expressed at suboptimal levels. Here, we describe an ensemble of mammalian protein expression vectors with CMV promoters of differing strengths. Using CHMP2A rescue of HIV-1 budding, we show that these vectors can combine high-transfection efficiencies with tunable protein expression levels to optimize the rescue of cellular phenotypes induced by siRNA transfection.     

Plant-driven weathering of apatite - the role of an ectomycorrhizal fungus

Ectomycorrhizal (EcM) fungi are increasingly recognized as important agents of mineral weathering and soil development, with far‐reaching impacts on biogeochemical cycles. Because EcM fungi live in a symbiotic relationship with trees and in close contact with bacteria and archaea, it is difficult to distinguish between the weathering effects of the fungus, host tree and other micro‐organisms. Here, we quantified mineral weathering by the fungus Paxillus involutus, growing in symbiosis with Pinus sylvestris under sterile conditions. The mycorrhizal trees were grown in specially designed sterile microcosms in which the supply of soluble phosphorus (P) in the bulk media was varied and grains of the calcium phosphate mineral apatite mixed with quartz, or quartz alone, were provided in plastic wells that were only accessed by their fungal partner. Under P limitation, pulse labelling of plants with ¹⁴CO₂ revealed plant‐to‐fungus allocation of photosynthates, with 17 times more ¹⁴C transferred into the apatite wells compared with wells with only quartz. Fungal colonization increased the release of P from apatite by almost a factor of three, from 7.5 (±1.1) × 10^?10 mol m^?2 s^?1 to 2.2 (±0.52) × 10^?9 mol m^?2 s^?1. On increasing the P supply in the microcosms from no added P, through apatite alone, to both apatite and orthophosphate, the proportion of biomass in roots progressively increased at the expense of the fungus. These three observations, (i) proportionately more plant energy investment in the fungal partner under P limitation, (ii) preferential fungal transport of photosynthate‐derived carbon towards patches of apatite grains and (iii) fungal enhancement of weathering rate, reveal the tightly coupled plant–fungal interactions underpinning enhanced EcM weathering of apatite and its utilization as P source.     

Evolution of trees and mycorrhizal fungi intensifies silicate mineral weathering

Forested ecosystems diversified more than 350 Ma to become major engines of continental silicate weathering, regulating the Earth''s atmospheric carbon dioxide concentration by driving calcium export into ocean carbonates. Our field experiments with mature trees demonstrate intensification of this weathering engine as tree lineages diversified in concert with their symbiotic mycorrhizal fungi. Preferential hyphal colonization of the calcium silicate-bearing rock, basalt, progressively increased with advancement from arbuscular mycorrhizal (AM) to later, independently evolved ectomycorrhizal (EM) fungi, and from gymnosperm to angiosperm hosts with both fungal groups. This led to ‘trenching’ of silicate mineral surfaces by AM and EM fungi, with EM gymnosperms and angiosperms releasing calcium from basalt at twice the rate of AM gymnosperms. Our findings indicate mycorrhiza-driven weathering may have originated hundreds of millions of years earlier than previously recognized and subsequently intensified with the evolution of trees and mycorrhizas to affect the Earth''s long-term CO₂ and climate history.     

Genome-Wide Association Studies of Quantitatively Measured Skin,Hair, and Eye Pigmentation in Four European Populations

Pigmentation of the skin, hair, and eyes varies both within and between human populations. Identifying the genes and alleles underlying this variation has been the goal of many candidate gene and several genome-wide association studies (GWAS). Most GWAS for pigmentary traits to date have been based on subjective phenotypes using categorical scales. But skin, hair, and eye pigmentation vary continuously. Here, we seek to characterize quantitative variation in these traits objectively and accurately and to determine their genetic basis. Objective and quantitative measures of skin, hair, and eye color were made using reflectance or digital spectroscopy in Europeans from Ireland, Poland, Italy, and Portugal. A GWAS was conducted for the three quantitative pigmentation phenotypes in 176 women across 313,763 SNP loci, and replication of the most significant associations was attempted in a sample of 294 European men and women from the same countries. We find that the pigmentation phenotypes are highly stratified along axes of European genetic differentiation. The country of sampling explains approximately 35% of the variation in skin pigmentation, 31% of the variation in hair pigmentation, and 40% of the variation in eye pigmentation. All three quantitative phenotypes are correlated with each other. In our two-stage association study, we reproduce the association of rs1667394 at the OCA2/HERC2 locus with eye color but we do not identify new genetic determinants of skin and hair pigmentation supporting the lack of major genes affecting skin and hair color variation within Europe and suggesting that not only careful phenotyping but also larger cohorts are required to understand the genetic architecture of these complex quantitative traits. Interestingly, we also see that in each of these four populations, men are more lightly pigmented in the unexposed skin of the inner arm than women, a fact that is underappreciated and may vary across the world.