Anion Carrier Research Articles

Polymorphisms in the uncoupling protein 3 gene and their associations with feed efficiency in chickens.

ObjectiveThe uncoupling protein 3 (UCP3) is a member of the mitochondrial anion carrier superfamily and has crucial effects on growth and feed efficiency in many species. Therefore, the objective of the present study was to examine the association of polymorphisms in the UCP3 gene with feed efficiency in meat-type chickens.MethodsSix single nucleotide polymorphisms (SNPs) of the UCP3 gene were chosen to be genotyped using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry in meat-type chicken populations with 724 birds in total. Body weight at 49 (BW49) and 70 days of age (BW70) and feed intake (FI) in the interval were collected, then body weight gain (BWG) and feed conversion ratio (FCR) were calculated individually.ResultsOne SNP with a low minor allele frequency (<1%) was removed by quality control and data filtering. The results showed that rs13997809 of UCP3 was significantly associated with BWG and FCR (p<0.05), and that rs13997811 had significant effects on BW70 and BWG (p<0.05). Rs13997812 of UCP3 was strongly associated with BW70, FI, and FCR (p<0.05). Furthermore, individuals with AA genotype of rs13997809 had significantly higher BWG and lower FCR (p<0.05) than those with AT genotype. The GG individuals showed strongly higher BW70 and BWG than AA birds in rs13997811 (p<0.05). Birds with the TT genotype of rs13997812 had significantly greater BW70 and lower FCR compared with the CT birds (p<0.05). In addition, the TAC haplotype based on rs13997809, rs13997811, and rs13997812 showed significant effects on BW70, FI, and FCR (p<0.05).ConclusionOur results therefore demonstrate important roles for UCP3 polymorphisms in growth and feed efficiency that might be used in meat-type chicken breeding programs.

Anion Recognition by a Bioactive Diureidodecalin Anionophore: Solid-State, Solution, and Computational Studies.

Recent work has identified a bis‐(p‐nitrophenyl)ureidodecalin anion carrier as a promising candidate for biomedical applications, showing good activity for chloride transport in cells yet almost no cytotoxicity. To underpin further development of this and related compounds, a detailed structural and binding investigation is reported. Crystal structures of the transporter as five solvates confirm the diaxial positioning of urea groups while revealing a degree of conformational flexibility. Structures of complexes with Cl−, Br−, NO3 −, SO4 2− and AcO−, supported by computational studies, show how the binding site can adapt to accommodate these anions. 1H NMR binding studies revealed exceptionally high affinities for anions in DMSO, decreasing in the order SO4 2−>H2PO4 −≈HCO3 −≈AcO−≫HSO4 −>Cl−>Br−>NO3 −>I−. Analysis of the binding results suggests that selectivity is determined mainly by the H‐bond acceptor strength of different anions, but is also modulated by receptor geometry.

Anthracene Bisureas as Powerful and Accessible Anion Carriers.

Synthetic anion carriers (anionophores) have potential as biomedical research tools and as treatments for conditions arising from defective natural transport systems (notably cystic fibrosis). Highly active anionophores that are readily accessible and easily deliverable are especially valuable. Previous work has resulted in steroid and trans‐decalin based anionophores with exceptional activity for chloride/nitrate exchange in vesicles, but poor accessibility and deliverability. This work shows that anthracene 1,8‐bisureas can fulfil all three criteria. In particular, a bis‐nitrophenyl derivative is prepared in two steps from commercial starting materials, yet shows comparable transport activity to the best currently known. Moreover, unlike earlier highly active systems, it does not need to be preincorporated in test vesicles but can be introduced subsequent to vesicle formation. This transporter also shows the ability to transfer between vesicles, and is therefore uniquely effective for anion transport at low transporter loadings. The results suggest that anthracene bisureas are promising candidates for application in biological research and medicine.

Lack of Uncoupling Protein 3 Protects from High‐Fat Diet‐Induced Insulin Resistance and Glucose Intolerance in Rats

Uncoupling protein 3 (UCP3), a mitochondrial anion carrier regulating fatty acid metabolism, is down‐regulated in type 2 diabetes. Whether UCP3 decrease influences the development of obesity and insulin resistance is currently unclear. We investigated the effect of genetically‐induced partial (UCP3+/−, with a 50% decrease in protein levels) or total (UCP3−/−) deficiency in whole body UCP3 on metabolic fitness in rats. Two lines of UCP3 deficient rats (hereinafter referred to as 1‐bpINS and 22‐bpDEL) were independently generated on a Sprague Dawley background strain using CRISPR‐Cas9 genome editing. Male UCP3 deficient rats and their wild type littermates were maintained on a chow diet or fed a high‐fat diet (HFD) for 14 weeks starting at 8 weeks of age. Metabolic fitness was assessed by analyzing body composition with EchoMRI and by performing insulin and glucose tolerance tests. Although both rat lines had similar lean‐to‐fat ratio and body mass under normal feeding conditions, the 22‐bpDEL line had higher circulating levels of lipids, insulin, and leptin, and was less insulin sensitive and glucose tolerant than the 1‐bpINS line. Partial or total UCP3 deficiency did not cause changes in body weight gain, body composition, or insulin sensitivity when rats were maintained on a chow diet. However, partial and total UCP3 deficiency increased glucose clearance at 15 min following intraperitoneal glucose injection in both rat lines. Compared to chow diet, high‐fat feeding significantly increased weight gain and fat mass while simultaneously impairing insulin sensitivity and glucose tolerance in both rat lines. In the 1‐bpINS line, loss of UCP3 decreased fat mass gain without altering food consumption so that UCP3−/− rats presented higher lean‐to‐fat ratio compared to wild type littermates. Loss of UCP3 improved insulin sensitivity in HFD‐fed 1‐bpINS rats, with a protective effect that was more prominent in UCP3−/− than in the UCP3+/− animals. Likewise, the gradual loss of UCP3 improved glucose tolerance in 1‐bpINS rats and prevented the increase of the glucose area under the curve (AUC) for UCP3−/− rats when compared to chow‐fed values (AUC 20219 vs. 15667 for wild type, P &lt; 0.0001; AUC 18724 vs. 15376 for UCP3+/−, P &lt; 0.01; AUC 17224 vs. 15007 for UCP3−/−, P = 0.4). This improvement of metabolic fitness associated with UCP3 deficiency was absent in rats from the 22‐bpDEL line. In conclusion, loss of UCP3 unexpectedly preserves metabolic fitness in male rats under high‐fat feeding. This protective effect depends on genetic background.Support or Funding InformationSupported by grants R00 HL112952, P01 HL051971, and P20 GM104357 from the National Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hetero‐Bambusurils

AbstractSynthetic anion carriers are essential for studying natural ion transporters and channels and for useful applications, such as treatment of channelopathies, supramolecular architecture, anion sensing and catalysis. Driven by the hypothesis that replacement of oxygen atoms in bambusurils (BUs) by other heteroatoms could significantly modify their anion binding properties, we calculated their molecular electrostatic potential and found a general trend of anion‐affinity: S&gt;O&gt;NH. We confirmed these predictions experimentally by synthesizing semithio‐ and semiaza‐BUs and studying their binding and transport properties. Although all analogs are excellent anion binders, only semithio‐bambus[6]uril is an effective transmembrane transporter capable of polarizing lipid membranes through selective anion uniport. Semiaza‐BUs exhibit simultaneous accommodation of three anions, linearly positioned along the main symmetry axis, which is reminiscent of natural chloride channels in E. coli.

Carrier-mediated solvent bar microextraction coupled with HPLC-DAD for the quantitative analysis of the hydrophilic antihypertensive peptide VLPVPR in human plasma

Aliquat-336, an anion carrier, facilitates the extraction of hydrophilic VLPVPR peptide by Solvent-Bar-Micro-Extraction, achieving sensitive peptide analysis in biological samples.

Full elucidation of the transmembrane anion transport mechanism of squaramides using in silico investigations.

A comprehensive experimental and theoretical investigation of the transmembrane chloride transport promoted by four series of squaramide derivatives, with different degrees of fluorination, number of convergent N-H binding units and conformational shapes, is reported. The experimental chloride binding and transport abilities of these small synthetic molecules in liposomes were rationalised with quantum descriptors and molecular dynamics simulations in POPC bilayers. The tripodal tren-based compounds, with three squaramide binding motifs, have high chloride affinity, isolating the anion from water molecules within the membrane model and preventing its release to the aqueous phase, in agreement with the absence of experimental transport activity. In contrast, the symmetrical mono-squaramides, with moderate chloride binding affinity, are able to bind and release chloride either in the aqueous phase or at the membrane interface level, in line with experimentally observed high transport activity. The PMF profiles associated with the diffusion of these free transporters and their chloride complexes across phospholipid bilayers show that the assisted chloride translocation is thermodynamically favoured.

Chemically modified (poly)vinyl chloride with built-in neutral carrier function as a new material for ion selective electrodes

The procedure for introducing 4-(trifluoroacetyl)benzoate groups into (poly)vinyl chloride has been developed. The new material thus prepared, 4-(trifluoroacetyl)benzoate modified (poly)vinyl chloride (TFAB-PVC), contains a built-in neutral anion carrier function. It has been shown that using TFAB-PVC instead of a known anion carrier additive, 4-(trifluoroacetyl)benzoic acid heptyl ester (TFABAHE), in sulfate, hydrogen phosphate and sulfide anion selective electrodes considerably increases the electrode’s lifetime even in alkaline media, probably by preventing crystallization of TFABAHE hydrate form on the membrane surface and slowing down the hydrolysis of ester groups and dissolution of the anion carrier.

Ion-selective electrodes based on long-chain quaternary ammonium salts with enhanced steric accessibility, and their application for determination of hydrophilic double-charged inorganic anion

The effect of long-chain quaternary ammonium salts’ exchange center steric accessibility and presence of a neutral anion carrier, 4-(trifluoroacetyl)benzoic acid heptyl ester, upon analytical characteristics of ion-selective electrodes reversible to molybdate, tungstate, sulfate, hydrogen phosphate, sulfite, carbonate and thiosulfate anions, has been studied. When the most sterically hindered quaternary ammonium salts, trinonyl octadecyl ammonium bromide, is replaced by one of the salts with the accessible exchange center, the selectivity is altered, the magnitude of the selectivity coefficient change being determined by the nature of the interfering ion. Thus, for carbonate- and sulfate-selective ISEs with hydrophobic thiocyanate ions as interference, the effect achieved is 6–8 orders of magnitude, while with nitrate ions as interference it is 3.4–5.7 orders, and for such hydrophilic interference ions as sulfate, chloride and oxalate, it is even less. Replacing trinonyl octadecyl ammonium bromide with a sterically accessible ammonium salt improves lower detection limit generally by 1–2 orders, while for sulfite and thiosulfate ISEs the effect is only 0.3–0.95 orders. The procedures for determination of sulfate, sulfite and carbonate ions in real objects (mineral water, dried fruits) have been proposed. They are simple, reasonably accurate, require only widely available equipment and can be recommended for use in chemical and food industries.

DFT study on the complexation of anions with 1,4,7,10,13,16-hexaazacyclooctodeca-2,5,8,11,14,17-hexaene

Macrocyclic compounds have been widely used as anion carriers, as they play important functions in chemical and biological systems. This work reports a theoretical study on free 1,4,7,10,13,16-hexaazacyclooctodeca-2,5,8,11,14,17-hexaene (HAC), as well as its complex with fluoride, chloride, bromide and acetate anions, with and without the presence of the sodium counterion, in the gas phase and implicit solvents (cyclohexane and acetonitrile), at the ωB97X-D/6-311G(d,p) level. The negative ∆G0 values indicate that the crown-anion complex is prone to be formed due to hydrogen bonds in all tested media. Nevertheless, such interactions weaken as the solvent polarity increases. The ΔG0 C6H12 values decrease when the counterion is taken into account, reinforcing the formation of the Na+‒HAC‒X− complex. However, the complexation is disfavored in polar solution, since the presence of the counterion increases the HAC-anion distance. Natural bond orbital analysis, the quantum theory of atoms in molecules and non-covalent interactions methods explored the nature and strength of the hydrogen bond interactions, while spin–spin coupling constant calculations for the fluoride-based complex (1h J F,H(N)) gave insight into the potential of this NMR parameter to experimentally probe the complexation of HAC with fluoride.

Pig Has No Brown Adipose Tissue

Brown adipose tissue (BAT) is critical for mammal's survival in the cold environment. UCP1 is responsible for the thermogenesis in the BAT and is recognized as a specific marker for BAT. Pig is a useful medical model for humans due to its similarity in size and physiology. Pig is also important economically as a meat‐producing livestock. A previous study identified brown adipocytes in different pig adipose tissues by microscopic and electron microscopic methods. In one immunoblotting study, UCP1 was not detected in any porcine adipose tissues by the rabbit anti‐rat UCP1 antibody. But, a recent study showed that UCP1 protein is present in porcine adipose tissue and is responsive to postnatal leptin treatment. These authors used an ovine UCP1 antibody in their immunoblotting assay. However, the positive result may be explained by non‐specific hybridization with other mitochondrial anion carrier proteins, such as UCP2 or UCP3 by the antibody used in the previous study. Therefore, whether BAT or more precisely UCP1 protein, exists in pig has never been conclusively established. The objective of this study was to ascertain whether pig has UCP1 protein.In this study, we used rapid amplification of cDNA ends (RACE) technique to obtain the UCP1 mRNA 3′ end sequence, confirmed only exons 1 and 2 of the UCP1 gene are transcribed in the pig. We then cloned the pig UCP1 gene exons 1 and 2, and used these two exons to express pig UCP1 protein by E. coli BL21. We purified and used the expressed pig UCP1 protein as antigen for antibody production in a rabbit. Immunoblotting assay was conducted using inguinal fat, interscapular fat, epididymal fat, retroperitoneal fat, supraclavicular fat, perirenal fat, neck fat, liver, muscle from pigs. Mice BAT was used as a positive control. Our rabbit anti‐pig UCP1 antibody can hybridize with mice UCP1 protein (about 35KD) and pig UCP1 (about 15KD) expressed by E. coli BL21, but could not detect UCP1 protein in any pig tissues. Our data clearly show only exons 1 and 2 of UCP1 gene are transcribed in pig adipose tissue, but the truncated pig UCP1 mRNA is not being translated. Since these results unequivocally demonstrate that pig has no UCP1 protein; therefore, the pig has no brown adipose tissue.Support or Funding InformationThis work was supported by the National High Technology Research and Development Program 863 (#2013AA102502); the National Natural Science Foundation of China (#31372283); The Team Project of Guangdong Agricultural Bureau (#2016LM2148) and the Natural Fund Key Projects of Guangdong Province (#2015A030311006). C.H. is supported by the USDA National Institute of Food and Agriculture, Hatch project HAW‐H2037, managed by the College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa.

Electrogenic transport by lipophilic guanidinium salts as anion carriers in bilayer membranes

Anion transport by four isomers of tetradecylguanidinium in vesicle bilayer membranes was investigated by ion-selective electrode and pH-sensitive fluorescence methods. On a macroscopic scale, these carriers mediate hydroxide-anion antiport. In the absence of an externally imposed ionic gradient, the carriers produce a pH gradient due to rapid partition-deprotonation of the carrier and diffusion of the guanidine conjugate base. The formally coupled anion transport is slow relative to this process, and the system exhibits a corresponding membrane polarisation detected with potential-sensitive fluorescent dyes. Selective anion transport in competition with the efficient hydroxide transport is detected only with relatively easily extracted anions and N,N′-disubstituted isomers. Membrane polarisation changes can be deduced from simple equivalent circuits derived from the Goldman–Hodgkin–Katz voltage equation.

Low concentration of uncouplers of oxidative phosphorylation decreases the TNF-induced endothelial permeability and lethality in mice

Low concentration of uncouplers of oxidative phosphorylation decreases the TNF-induced endothelial permeability and lethality in mice

Bambusurils as effective ion caging agents: Does desolvation guide conformation?

Bambusurils as effective ion caging agents: Does desolvation guide conformation?

Mitochondrial Uncoupling Carrier protein -2 DNA polymorphism (−866 G/A) as a risk for polycystic ovary Syndrome in King Saudi Arabian Females

PCOS (Polycystic ovary syndrome) is a common hormonal disorder characterized by gonadotropin dysregulation, hyperandrogenism, menstrual irregularity and ovarian morphology. The uncoupling protein-2 (UCP-2) gene is a member of the MACP (mitochondrial anion carrier protein) and it is strongly associated with insulin resist and obesity. Aim of the work: to detect the association between PCOS and -866G/A polymorphism in UCP2 gene promoter. Subjects and methods: The study include Forty-seven patients with polycystic ovary syndrome and fifty–six controls. Blood samples were taken for biochemical tests and Restriction fragment length polymorphism (RFLP) for detection of -866 G/A UCP2 gene polymorphisms. Results and conclusion : Significant associations between the -866A/A -866G/A of UCP2 gene promotor and PCOS were found. In addition, a Significant association between -866A/A polymorphism of UCP2 and both obesity (high BMI) and type 2 diabetes in the same study groups.

Association and expression analyses of the Ucp2 and Ucp3 gene polymorphisms with body measurement and meat quality traits in Qinchuan cattle.

The uncoupling proteins (UCPs) belong to the mitochondrial inner membrane anion carrier superfamily and play an important role in energy homeostasis. Genetic studies have demonstrated that Ucp2 and Ucp3 gene variants are involved in obesity and metabolic syndrome. The aim of this study was to identify associations between polymorphisms of Ucp2 and Ucp3 genes and economically-important traits in Qinchuan cattle. In the present study, one single-nucleotide polymorphism (SNP) in the 5'UTR region (SNP1:g.C-754G) of the Ucp2 gene was identified by direct sequencing of 441 Qinchuan cattle. Two SNPs in exon 3 (SNP2: g.G4877A: SNP3: g.C4902T) of the Ucp3 gene were identified by sequencing and polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) among 441 Qinchuan cattle. Association analysis showed that SNP1 and SNP2 were associated with the meat quality traits (MQTs) including back fat thickness, loin muscle area and intramuscular fat content. SNP3 was found to be associated with part of the body measurement traits (BMTs) which referred to withers height and chest depth. In addition, QTL pyramiding analysis showed that individuals with diplotype P3P3 (GG-GG-CC) exhibited the best performance in terms of back fat thickness, loin muscle area, intramuscular fat content, rump length, hip width, chest depth and chest circumference. With regard to the G4877A mutation, real time PCR analysis revealed that individuals with AA genotype of the Ucp3 gene expressed higher mRNA levels than those with GG genotype. These results suggest that the diplotype P3P3 (GG-GG-CC) could be used as a molecular marker of the combined genotypes for future selection of body measurement traits and meat quality traits in Qinchuan cattle.

The Effect of Resveratrol and Quercetin Treatment on PPAR Mediated Uncoupling Protein (UCP-) 1, 2, and 3 Expression in Visceral White Adipose Tissue from Metabolic Syndrome Rats.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily involved in the control of body temperature and energy balance regulation. They are currently proposed as therapeutic targets for treating obesity and metabolic syndrome (MetS). We studied the gene expression regulation of UCP1, -2, and -3 in abdominal white adipose tissue (WAT) from control and MetS rats treated with two doses of a commercial mixture of resveratrol (RSV) and quercetin (QRC). We found that UCP2 was the predominantly expressed isoform, UCP3 was present at very low levels, and UCP1 was undetectable. The treatment with RSV + QRC did not modify UCP3 levels; however, it significantly increased UCP2 mRNA in control and MetS rats in association with an increase in oleic and linoleic fatty acids. WAT from MetS rats showed a significantly increased expression of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ when compared to the control group. Furthermore, PPAR-α protein levels were increased by the highest dose of RSV + QRC in the control and MetS groups. PPAR-γ expression was only increased in the control group. We conclude that the RSV + QRC treatment leads to overexpression of UCP2, which is associated with an increase in MUFA and PUFA, which might increase PPAR-α expression.

PH-Regulated Nonelectrogenic Anion Transport by Phenylthiosemicarbazones.

Gated ion transport across biological membranes is an intrinsic process regulated by protein channels. Synthetic anion carriers (anionophores) have potential applications in biological research; however, previously reported examples are mostly nonspecific, capable of mediating both electrogenic and electroneutral (nonelectrogenic) transport processes. Here we show the transmembrane Cl(-) transport studies of synthetic phenylthiosemicarbazones mimicking the function of acid-sensing (proton-gated) ion channels. These anionophores have remarkable pH-switchable transport properties with up to 640-fold increase in transport efficacy on going from pH 7.2 to 4.0. This "gated" process is triggered by protonation of the imino nitrogen and concomitant conformational change of the anion-binding thiourea moiety from anti to syn. By using a combination of two cationophore-coupled transport assays, with either monensin or valinomycin, we have elucidated the fundamental transport mechanism of phenylthiosemicarbazones which is shown to be nonelectrogenic, inseparable H(+)/Cl(-) cotransport. This study demonstrates the first examples of pH-switchable nonelectrogenic anion transporters.

Tilting and Tumbling in Transmembrane Anion Carriers: Activity Tuning through n-Alkyl Substitution.

Anion transport by synthetic carriers (anionophores) holds promise for medical applications, especially the treatment of cystic fibrosis. Among the factors which determine carrier activity, the size and disposition of alkyl groups is proving remarkably important. Herein we describe a series of dithioureidodecalin anionophores, in which alkyl substituents on one face are varied from C0 to C10 in two‐carbon steps. Activities increase then decrease as the chain length grows, peaking quite sharply at C6. Molecular dynamics simulations showed the transporter chloride complexes releasing chloride as they approach the membrane‐aqueous interface. The free transporter then stays at the interface, adopting an orientation that depends on the alkyl substituent. If chloride release is prevented, the complex is positioned similarly. Longer chains tilt the binding site away from the interface, potentially freeing the transporter or complex to move through the membrane. However, chains which are too long can also slow transport by inhibiting movement, and especially reorientation, within the phospholipid bilayer.

Efficient, non-toxic anion transport by synthetic carriers in cells and epithelia.

Transmembrane anion transporters (anionophores) have potential for new modes of biological activity, including therapeutic applications. In particular they might replace the activity of defective anion channels in conditions such as cystic fibrosis. However, data on the biological effects of anionophores are scarce, and it remains uncertain whether such molecules are fundamentally toxic. Here, we report a biological study of an extensive series of powerful anion carriers. Fifteen anionophores were assayed in single cells by monitoring anion transport in real time through fluorescence emission from halide-sensitive yellow fluorescent protein. A bis-(p-nitrophenyl)ureidodecalin shows especially promising activity, including deliverability, potency and persistence. Electrophysiological tests show strong effects in epithelia, close to those of natural anion channels. Toxicity assays yield negative results in three cell lines, suggesting that promotion of anion transport may not be deleterious to cells. We therefore conclude that synthetic anion carriers are realistic candidates for further investigation as treatments for cystic fibrosis.