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1.
J Biol Chem ; 2020 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-32161117

RESUMO

The canonical pathway of eicosanoid production in most mammalian cells is initiated by phospholipase A2-mediated release of arachidonic acid, followed by its enzymatic oxidation resulting in a vast array of eicosanoid products. However, recent work has demonstrated that the major phospholipase in mitochondria, iPLA2γ (patatin-like phospholipase domain containing 8 [PNPLA8]), possesses sn-1 specificity, with polyunsaturated fatty acids at the sn-2 position generating polyunsaturated sn-2-acyl lysophospholipids. Through strategic chemical derivatization, chiral chromatographic separation, and multistage tandem MS, here we first demonstrate that human platelet-type 12-lipoxygenase (12-LOX) can directly catalyze the regioselective and stereospecific oxidation of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) and 2-arachidonoyl-lysophosphatidylethanolamine (2-AA-LPE). Next, we identified these two eicosanoid-lysophospholipids in murine myocardium and in isolated platelets. Moreover, we observed robust increases in 2-AA-LPC, 2-AA-LPE, and their downstream 12-LOX oxidation products, 12(S)-HETE-LPC and 12(S)-HETE-LPE, in calcium ionophore (A23187)-stimulated murine platelets. Mechanistically, genetic ablation of iPLA2γ markedly decreased the calcium-stimulated production of 2-AA-LPC, 2-AA-LPE, and 12-HETE-lysophospholipids in mouse platelets. Importantly, a potent and selective 12-LOX inhibitor, ML355, significantly inhibited the production of 12-HETE-LPC and 12-HETE-LPE in activated platelets. Furthermore, we found that aging is accompanied by significant changes in 12-HETE-LPC in murine serum that were also markedly attenuated by the iPLA2γ genetic ablation. Collectively, these results identify previously unknown iPLA2γ-initiated signaling pathways mediated by direct 12-LOX oxidation of 2-AALPC and 2-AA-LPE. This oxidation generates previously unrecognized eicosanoid-lysophospholipids that may serve as biomarkers for age-related diseases and could potentially be used as targets in therapeutic interventions.

2.
PLoS Genet ; 16(1): e1008527, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31999692

RESUMO

A form of hereditary cerebellar ataxia has recently been described in the Norwegian Buhund dog breed. This study aimed to identify the genetic cause of the disease. Whole-genome sequencing of two Norwegian Buhund siblings diagnosed with progressive cerebellar ataxia was carried out, and sequences compared with 405 whole genome sequences of dogs of other breeds to filter benign common variants. Nine variants predicted to be deleterious segregated among the genomes in concordance with an autosomal recessive mode of inheritance, only one of which segregated within the breed when genotyped in additional Norwegian Buhunds. In total this variant was assessed in 802 whole genome sequences, and genotyped in an additional 505 unaffected dogs (including 146 Buhunds), and only four affected Norwegian Buhunds were homozygous for the variant. The variant identified, a T to C single nucleotide polymorphism (SNP) (NC_006585.3:g.88890674T>C), is predicted to cause a tryptophan to arginine substitution in a highly conserved region of the potassium voltage-gated channel interacting protein KCNIP4. This gene has not been implicated previously in hereditary ataxia in any species. Evaluation of KCNIP4 protein expression through western blot and immunohistochemical analysis using cerebellum tissue of affected and control dogs demonstrated that the mutation causes a dramatic reduction of KCNIP4 protein expression. The expression of alternative KCNIP4 transcripts within the canine cerebellum, and regional differences in KCNIP4 protein expression, were characterised through RT-PCR and immunohistochemistry respectively. The voltage-gated potassium channel protein KCND3 has previously been implicated in spinocerebellar ataxia, and our findings suggest that the Kv4 channel complex KCNIP accessory subunits also have an essential role in voltage-gated potassium channel function in the cerebellum and should be investigated as potential candidate genes for cerebellar ataxia in future studies in other species.

3.
Vet Rec ; 185(13): 411, 2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31582496
4.
Sci Rep ; 9(1): 11060, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31363151

RESUMO

Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B. pseudomallei virulence and dormancy. The peptidoglycan remodeling enzymes, lytic transglycosylases (Ltgs) are potential targets for such new antibiotics. Ltgs cleave the glycosidic bonds within bacterial peptidoglycan allowing for the insertion of peptidoglycan precursors during cell growth and division, and cell membrane spanning structures such as flagella and secretion systems. Using bioinformatic analysis we have identified 8 putative Ltgs in B. pseudomallei K96243. We aimed to investigate one of these Ltgs, LtgG (BPSL3046) through the generation of deletion mutants and biochemical analysis. We have shown that LtgG is a key contributor to cellular morphology, division, motility and virulence in BALB/c mice. We have determined the crystal structure of LtgG and have identified various amino acids likely to be important in peptidoglycan binding and catalytic activity. Recombinant protein assays and complementation studies using LtgG containing a site directed mutation in aspartate 343, confirmed the essentiality of this amino acid in the function of LtgG.

5.
Genes (Basel) ; 10(6)2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31181696

RESUMO

Two FGF4 retrogenes on chromosomes 12 (12-FGF4RG) and 18 (18-FGF4RG) contribute to short-limbed phenotypes in dogs. 12-FGF4RG has also been associated with intervertebral disc disease (IVDD). Both of these retrogenes were found to be widespread among dog breeds with allele frequencies ranging from 0.02 to 1; however, their additive contribution to disease is unknown. Surgical cases of IVDD (n = 569) were evaluated for age of onset, disc calcification, and genotypes for the FGF4 retrogenes. Multivariable linear regression analysis identified the presence of one or two copies of 12-FGF4RG associated with significantly younger age at first surgery in a dominant manner. 18-FGF4RG had only a minor effect in dogs with one copy. Multivariable logistic regression showed that 12-FGF4RG had an additive effect on radiographic disc calcification, while 18-FGF4RG had no effect. Multivariable logistic regression using mixed breed cases and controls identified only 12-FGF4RG as highly associated with disc herniation in a dominant manner (Odds Ratio, OR, 18.42, 95% Confidence Interval (CI) 7.44 to 50.26; P < 0.001). The relative risk for disc surgery associated with 12-FGF4RG varied from 5.5 to 15.1 within segregating breeds and mixed breeds. The FGF4 retrogene on CFA12 acts in a dominant manner to decrease the age of onset and increase the overall risk of disc disease in dogs. Other modifiers of risk may be present within certain breeds, including the FGF4 retrogene on CFA18.


Assuntos
Doenças do Cão/genética , Fator 4 de Crescimento de Fibroblastos/genética , Predisposição Genética para Doença , Degeneração do Disco Intervertebral/genética , Deslocamento do Disco Intervertebral/genética , Animais , Cruzamento , Doenças do Cão/fisiopatologia , Cães , Frequência do Gene , Genótipo , Disco Intervertebral/fisiopatologia , Degeneração do Disco Intervertebral/fisiopatologia , Deslocamento do Disco Intervertebral/fisiopatologia , Fenótipo
6.
J Biol Chem ; 294(26): 10146-10159, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31080170

RESUMO

Recently, oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets. The canonical pathway for the synthesis of oxidized phospholipids is through the release of arachidonic acid by cytosolic phospholipase A2α (cPLA2α) followed by its enzymatic oxidation, activation of the carboxylate anion by acyl-CoA synthetase(s), and re-esterification to the sn-2 position by sn-2 acyltransferase activity (i.e. the Lands cycle). However, recent studies have demonstrated the unanticipated significance of sn-1 hydrolysis of arachidonoyl-containing choline and ethanolamine glycerophospholipids by other phospholipases to generate the corresponding 2-arachidonoyl-lysolipids. Herein, we identified a pathway for oxidized phospholipid synthesis comprising sequential sn-1 hydrolysis by a phospholipase A1 (e.g. by patatin-like phospholipase domain-containing 8 (PNPLA8)), direct enzymatic oxidation of the resultant 2-arachidonoyl-lysophospholipids, and the esterification of oxidized 2-arachidonoyl-lysophospholipids by acyl-CoA-dependent sn-1 acyltransferase(s). To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (d- and l-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring l-enantiomer of the ether analog of lysophosphatidylcholine. Next, we demonstrated the high selectivity of the sn-1 acyltransferase activity for saturated acyl-CoA species. Importantly, we established that 2-15-hydroxyeicosatetraenoic acid (HETE) ether-LPC sn-1 esterification is markedly activated by thrombin treatment of murine platelets to generate oxidized PC. Collectively, these findings demonstrate the enantiomeric specificity and saturated acyl-CoA selectivity of microsomal sn-1 acyltransferase(s) and reveal its participation in a previously uncharacterized pathway for the synthesis of oxidized phospholipids with cell-signaling properties.


Assuntos
Aciltransferases/metabolismo , Ácidos Hidroxieicosatetraenoicos/metabolismo , Lisofosfolipídeos/metabolismo , Fosfolipases/metabolismo , Fosfolipídeos/metabolismo , Acilação , Aciltransferases/genética , Animais , Plaquetas/metabolismo , Ácidos Hidroxieicosatetraenoicos/química , Lisofosfolipídeos/química , Camundongos , Microssomos Hepáticos/metabolismo , Oxirredução , Fosfolipídeos/química , Especificidade por Substrato
7.
PLoS One ; 14(3): e0214439, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30913266

RESUMO

Accurate species delimitation and description are necessary to guide effective conservation of imperiled species, and this synergy is maximized when multiple data sources are used to delimit species. We illustrate this point by examining Drymarchon couperi (Eastern Indigo Snake), a large, federally-protected species in North America that was recently divided into two species based on gene sequence data from three loci and heuristic morphological assessment. Here, we re-evaluate the two-species hypothesis for D. couperi by evaluating both population genetic and gene sequence data. Our analyses of 14 microsatellite markers revealed 6-8 genetic population clusters with significant admixture, particularly across the contact zone between the two hypothesized species. Phylogenetic analyses of gene sequence data with maximum-likelihood methods suggested discordance between mitochondrial and nuclear markers and provided phylogenetic support for one species rather than two. For these reasons, we place Drymarchon kolpobasileus into synonymy with D. couperi. We suggest inconsistent patterns between mitochondrial and nuclear DNA are driven by high dispersal of males relative to females. We advocate for species delimitation exercises that evaluate admixture and gene flow in addition to phylogenetic analyses, particularly when the latter reveal monophyletic lineages. This is particularly important for taxa, such as squamates, that exhibit strong sex-biased dispersal. Problems associated with over-delimitation of species richness can become particularly acute for threatened and endangered species, because of high costs to conservation when taxonomy demands protection of more individual species than are supported by accumulating data.


Assuntos
Distribuição Animal , Núcleo Celular/genética , Conservação dos Recursos Naturais , Espécies em Perigo de Extinção , Mitocôndrias/genética , Serpentes/classificação , Serpentes/genética , Animais , Feminino , Loci Gênicos/genética , Masculino , Repetições de Microssatélites/genética , Fatores Sexuais
8.
J Am Vet Med Assoc ; 253(6): 774-780, 2018 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-30179085

RESUMO

CASE DESCRIPTION Two 12-week-old Norwegian Buhunds from a litter of 5 were evaluated because of slowly progressive cerebellar ataxia and fine head tremors. Two other females from the same pedigree had been previously evaluated for similar signs. CLINICAL FINDINGS Findings of general physical examination, CBC, and serum biochemical analysis were unremarkable for all affected puppies. Brain MRI and CSF analysis, including PCR assays for detection of Toxoplasma gondii, Neospora caninum, and canine distemper virus, were performed for 3 dogs, yielding unremarkable results. Urinary organic acid screening, enzyme analysis of fibroblasts cultured from skin biopsy specimens, and brainstem auditory-evoked response testing were performed for 2 puppies, and results were also unremarkable. TREATMENT AND OUTCOME The affected puppies were euthanized at the breeder's request, and their brains and spinal cords were submitted for histologic examination. Histopathologic findings included a markedly reduced expression of calbindin D28K and inositol triphosphate receptor 1 by Purkinje cells, with only mild signs of neuronal degeneration. Results of pedigree analysis suggested an autosomal recessive mode of inheritance. Candidate-gene analysis via mRNA sequencing for 2 of the affected puppies revealed no genetic variants that could be causally associated with the observed abnormalities. CLINICAL RELEVANCE Findings for the dogs of this report suggested the existence of a hereditary form of ataxia in Norwegian Buhunds with histologic characteristics suggestive of Purkinje cell dysfunction. The presence of hereditary ataxia in this breed must be considered both in clinical settings and for breeding strategies.


Assuntos
Doenças do Cão/patologia , Degenerações Espinocerebelares/veterinária , Animais , Diagnóstico Diferencial , Cães , Feminino , Masculino , Linhagem , Degenerações Espinocerebelares/patologia
9.
Ecol Evol ; 8(15): 7673-7687, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30151181

RESUMO

Standardized and repeatable data acquisition and analyses are required to enable the mapping and condition monitoring of reefs within Marine Protected Areas (MPAs). Changes in habitat condition must be reliably identified and reported to best support evidence-based management. Biogenic reefs in temperate waters, that is, hard matter created by living organisms and raised above the seabed, provide food and shelter for many plant and animal species. This article explores the feasibility of habitat mapping, using remote sensing datasets, as well as metrics for repeatable and suitable assessment of areas of Sabellaria spinulosa for their status as biogenic reef. Data were gathered within the North Norfolk Sandbanks and Saturn Reef candidate Special Area of Conservation/Site of Community Importance in the southern North Sea. Six study areas were identified as potential locations of biogenic reef using previously acquired data, and these were targeted for further investigation using a combination of high resolution multibeam echosounder and sidescan sonar. Where potential S. spinulosa was identified from the acoustic data, a drop-down camera system was employed for visual verification. Areas of known and potential S. spinulosa reef were mapped successfully at two of the six study areas, although future approaches should take careful consideration of the seabed morphology and predominant habitat backdrop to successfully interpret such data. Camera tows from S. spinulosa reef areas were broken up into 5-s segments, with each segment scored for (a) average tube elevation; (b) average percentage cover; and (c) for the presence or absence of S. spinulosa. These metrics were utilized to create summary statistics, including a value of patchiness derived from presence/absence data, that is recommended for application as part of future monitoring programs. The application of this methodology could benefit wider assessments of similar threated or declining habitats such as intertidal Mytilus edulis beds on mixed and sandy sediments, Maerl beds, Modioulus modiolus beds, Ostrea edulis beds, and Zostera beds where patchiness may also be considered of environmental importance.

10.
Med Phys ; 45(6): 2476-2485, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29635795

RESUMO

PURPOSE: The aim of this work was to create tissue-mimicking gel phantoms appropriate for diffusion kurtosis imaging (DKI) for quality assurance, protocol optimization, and sequence development. METHODS: A range of agar, agarose, and polyvinyl alcohol phantoms with concentrations ranging from 1.0% to 3.5%, 0.5% to 3.0%, and 10% to 20%, respectively, and up to 3 g of glass microspheres per 100 ml were created. Diffusion coefficients, excess kurtosis values, and relaxation rates were experimentally determined. RESULTS: The kurtosis values for the plain gels ranged from 0.05 with 95% confidence interval (CI) of (0.029,0.071) to 0.216(0.185,0.246), well below the kurtosis values reported in the literature for various tissues. The addition of glass microspheres increased the kurtosis of the gels with values up to 0.523(0.465,0.581) observed for gels with the highest concentration of microspheres. Repeat scans of some of the gels after more than 6 months of storage at room temperature indicate changes in the diffusion parameters of less than 10%. The addition of the glass microspheres reduces the apparent diffusion coefficients (ADCs) and increases the longitudinal and transverse relaxation rates, but the values remain comparable to those for plain gels and tissue, with ADCs observed ranging from 818(585,1053) × 10-6  mm2 /s to 2257(2118,2296) × 10-6  mm2 /s, R1 values ranging from 0.34(0.32,0.35) 1/s to 0.51(0.50,0.52) 1/s, and R2 values ranging from 9.69(9.34,10.04) 1/s to 33.07(27.10, 39.04) 1/s. CONCLUSIONS: Glass microspheres can be used to effectively modify diffusion properties of gel phantoms and achieve a range of kurtosis values comparable to those reported for a variety of tissues.


Assuntos
Imagem de Difusão por Ressonância Magnética/instrumentação , Modelos Anatômicos , Imagens de Fantasmas , Ágar , Imagem de Difusão por Ressonância Magnética/métodos , Desenho de Equipamento , Géis , Vidro , Humanos , Microesferas , Álcool de Polivinil , Garantia da Qualidade dos Cuidados de Saúde , Sefarose , Água
11.
J Biol Chem ; 293(22): 8693-8709, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29530984

RESUMO

Plasmalogens are phospholipids critical for cell function and signaling that contain a vinyl ether linkage at the sn-1 position and are highly enriched in arachidonic acid (AA) at the sn-2 position. However, the enzyme(s) responsible for the cleavage of the vinyl ether linkage in plasmalogens has remained elusive. Herein, we report that cytochrome c, in the presence of either cardiolipin (CL), O2 and H2O2, or oxidized CL and O2, catalyzes the oxidation of the plasmalogen vinyl ether linkage, promoting its hydrolytic cleavage and resultant production of 2-AA-lysolipids and highly reactive α-hydroxy fatty aldehydes. Using stable isotope labeling in synergy with strategic chemical derivatizations and high-mass-accuracy MS, we deduced the chemical mechanism underlying this long sought-after reaction. Specifically, labeling with either 18O2 or H218O, but not with H218O2, resulted in M + 2 isotopologues of the α-hydroxyaldehyde, whereas reactions with both 18O2 and H218O identified the M + 4 isotopologue. Furthermore, incorporation of 18O from 18O2 was predominantly located at the α-carbon. In contrast, reactions with H218O yielded 18O linked to the aldehyde carbon. Importantly, no significant labeling of 2-AA-lysolipids with 18O2, H218O, or H218O2 was present. Intriguingly, phosphatidylinositol phosphates (PIP2 and PIP3) effectively substituted for cardiolipin. Moreover, cytochrome c released from myocardial mitochondria subjected to oxidative stress cleaved plasmenylcholine in membrane bilayers, and this was blocked with a specific mAb against cytochrome c Collectively, these results identify the first plasmalogenase in biology, reveal the production of previously unanticipated signaling lipids by cytochrome c, and present new perspectives on cellular signaling during oxidative stress.


Assuntos
Citocromos c/metabolismo , Hidrolases/metabolismo , Mitocôndrias Cardíacas/metabolismo , Estresse Oxidativo , Plasmalogênios/metabolismo , Compostos de Vinila/química , Animais , Citocromos c/química , Cavalos , Humanos , Hidrólise , Lipídeos/análise , Masculino , Oxirredução , Coelhos , Compostos de Vinila/metabolismo
12.
Nat Commun ; 9(1): 765, 2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29472584

RESUMO

Calcium-independent phospholipase A2ß (iPLA2ß) regulates important physiological processes including inflammation, calcium homeostasis and apoptosis. It is genetically linked to neurodegenerative disorders including Parkinson's disease. Despite its known enzymatic activity, the mechanisms underlying iPLA2ß-induced pathologic phenotypes remain poorly understood. Here, we present a crystal structure of iPLA2ß that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. They are surrounded by ankyrin repeat domains that adopt an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. The structure and additional solution studies suggest that both catalytic domains can be bound and allosterically inhibited by a single calmodulin. These features suggest mechanisms of iPLA2ß cellular localization and activity regulation, providing a basis for inhibitor development. Furthermore, the structure provides a framework to investigate the role of neurodegenerative mutations and the function of iPLA2ß in the brain.


Assuntos
Fosfolipases A2 do Grupo VI/química , Fosfolipases A2 do Grupo VI/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Domínio Catalítico , Cristalização , Dimerização , Regulação da Expressão Gênica , Fosfolipases A2 do Grupo VI/genética , Humanos , Ligação Proteica , Transporte Proteico
13.
Infect Immun ; 86(5)2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29440370

RESUMO

The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei A panel of B. pseudomalleidsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.


Assuntos
Burkholderia pseudomallei/genética , Burkholderia pseudomallei/patogenicidade , Melioidose/patologia , Proteínas de Membrana/imunologia , Camundongos Endogâmicos BALB C/imunologia , Oxirredutases/imunologia , Virulência/genética , Animais , Austrália , Burkholderia pseudomallei/imunologia , Modelos Animais de Doenças , Melioidose/genética , Melioidose/microbiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Oxirredutases/genética , Oxirredutases/metabolismo , Virulência/imunologia
14.
Genome Biol ; 19(1): 22, 2018 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-29452591

RESUMO

BACKGROUND: Mammalian species exhibit a wide range of lifespans. To date, a robust and dynamic molecular readout of these lifespan differences has not yet been identified. Recent studies have established the existence of ageing-associated differentially methylated positions (aDMPs) in human and mouse. These are CpG sites at which DNA methylation dynamics show significant correlations with age. We hypothesise that aDMPs are pan-mammalian and are a dynamic molecular readout of lifespan variation among different mammalian species. RESULTS: A large-scale integrated analysis of aDMPs in six different mammals reveals a strong negative relationship between rate of change of methylation levels at aDMPs and lifespan. This relationship also holds when comparing two different dog breeds with known differences in lifespans. In an ageing cohort of aneuploid mice carrying a complete copy of human chromosome 21, aDMPs accumulate far more rapidly than is seen in human tissues, revealing that DNA methylation at aDMP sites is largely shaped by the nuclear trans-environment and represents a robust molecular readout of the ageing cellular milieu. CONCLUSIONS: Overall, we define the first dynamic molecular readout of lifespan differences among mammalian species and propose that aDMPs will be an invaluable molecular tool for future evolutionary and mechanistic studies aimed at understanding the biological factors that determine lifespan in mammals.


Assuntos
Metilação de DNA , Longevidade/genética , Mamíferos/genética , Envelhecimento/genética , Animais , Cães , Humanos , Camundongos
15.
J Biol Chem ; 293(1): 115-129, 2018 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-29158256

RESUMO

Congestive heart failure typically arises from cardiac myocyte necrosis/apoptosis, associated with the pathological opening of the mitochondrial permeability transition pore (mPTP). mPTP opening decreases the mitochondrial membrane potential leading to the activation of Ca2+-independent phospholipase A2γ (iPLA2γ) and the production of downstream toxic metabolites. However, the array of enzymatic mediators and the exact chemical mechanisms responsible for modulating myocardial mPTP opening remain unclear. Herein, we demonstrate that human heart failure activates specific myocardial mitochondrial phospholipases that increase Ca2+-dependent production of toxic hydroxyeicosatetraenoic acids (HETEs) and attenuate the activity of phospholipases that promote the synthesis of protective epoxyeicosatrienoic acids (EETs). Mechanistically, HETEs activated the Ca2+-induced opening of the mPTP in failing human myocardium, and the highly selective pharmacological blockade of either iPLA2γ or lipoxygenases attenuated mPTP opening in failing hearts. In contrast, pharmacological inhibition of cytochrome P450 epoxygenases opened the myocardial mPTP in human heart mitochondria. Remarkably, the major mitochondrial phospholipase responsible for Ca2+-activated release of arachidonic acid (AA) in mitochondria from non-failing hearts was calcium-dependent phospholipase A2ζ (cPLA2ζ) identified by sequential column chromatographies and activity-based protein profiling. In contrast, iPLA2γ predominated in failing human myocardium. Stable isotope kinetics revealed that in non-failing human hearts, cPLA2ζ metabolically channels arachidonic acid into EETs, whereas in failing hearts, increased iPLA2γ activity channels AA into toxic HETEs. These results mechanistically identify the sequelae of pathological remodeling of human mitochondrial phospholipases in failing myocardium. This remodeling metabolically channels AA into toxic HETEs promoting mPTP opening, which induces necrosis/apoptosis leading to further progression of heart failure.


Assuntos
Fosfolipases A2 do Grupo VI/metabolismo , Insuficiência Cardíaca/metabolismo , Ácidos Hidroxieicosatetraenoicos/biossíntese , Mitocôndrias Cardíacas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/patologia , Humanos , Ácidos Hidroxieicosatetraenoicos/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/enzimologia , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/metabolismo , Miocárdio/enzimologia , Miocárdio/metabolismo , Miocárdio/patologia , Permeabilidade , Fosfolipases A2/metabolismo
16.
Am J Vet Res ; 79(1): 98-106, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29287154

RESUMO

OBJECTIVE To evaluate the coding regions of ADAMTS17 for potential mutations in Chinese Shar-Pei with a diagnosis of primary open-angle glaucoma (POAG), primary lens luxation (PLL), or both. ANIMALS 63 Shar-Pei and 96 dogs of other breeds. PROCEDURES ADAMTS17 exon resequencing was performed on buccal mucosal DNA from 10 Shar-Pei with a diagnosis of POAG, PLL, or both (affected dogs). A candidate causal variant sequence was identified, and additional dogs (53 Shar-Pei [11 affected and 42 unaffected] and 95 dogs of other breeds) were genotyped for the variant sequence by amplified fragment length polymorphism analysis. Total RNA was extracted from ocular tissues of 1 affected Shar-Pei and 1 ophthalmologically normal Golden Retriever; ADAMTS17 cDNA was reverse transcribed and sequenced, and ADAMTS17 expression was evaluated by quantitative reverse-transcription PCR assay. RESULTS All affected Shar-Pei were homozygous for a 6-bp deletion in exon 22 of ADAMTS17 predicted to affect the resultant protein. All unaffected Shar-Pei were heterozygous or homozygous for the wild-type allele. The variant sequence was significantly associated with affected status (diagnosis of POAG, PLL, or both). All dogs of other breeds were homozygous for the wild-type allele. The cDNA sequencing confirmed presence of the expected variant mRNA sequence in ocular tissue from the affected dog only. Gene expression analysis revealed a 4.24-fold decrease in the expression of ADAMTS17 in ocular tissue from the affected dog. CONCLUSIONS AND CLINICAL RELEVANCE Results supported that the phenotype (diagnosis of POAG, PLL, or both) is an autosomal recessive trait in Shar-Pei significantly associated with the identified mutation in ADAMTS17.


Assuntos
Proteínas ADAMTS/genética , Doenças do Cão/genética , Glaucoma de Ângulo Aberto/veterinária , Subluxação do Cristalino/veterinária , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados/veterinária , Animais , Cruzamento , Cães , Feminino , Genótipo , Glaucoma de Ângulo Aberto/genética , Subluxação do Cristalino/genética , Masculino , Mutação , Fenótipo
17.
J Biol Chem ; 292(25): 10672-10684, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28442572

RESUMO

Cardiolipin (CL) is a dimeric phospholipid with critical roles in mitochondrial bioenergetics and signaling. Recently, inhibition of the release of oxidized fatty acyl chains from CL by the calcium-independent phospholipase A2γ (iPLA2γ)-selective inhibitor (R)-BEL suggested that iPLA2γ is responsible for the hydrolysis of oxidized CL and subsequent signaling mediated by the released oxidized fatty acids. However, chemical inhibition by BEL is subject to off-target pharmacologic effects. Accordingly, to unambiguously determine the role of iPLA2γ in the hydrolysis of oxidized CL, we compared alterations in oxidized CLs and the release of oxidized aliphatic chains from CL in experiments with purified recombinant iPLA2γ, germ-line iPLA2γ-/- mice, cardiac myocyte-specific iPLA2γ transgenic mice, and wild-type mice. Using charge-switch high mass accuracy LC-MS/MS with selected reaction monitoring and product ion accurate masses, we demonstrated that iPLA2γ is the major enzyme responsible for the release of oxidized aliphatic chains from CL. Our results also indicated that iPLA2γ selectively hydrolyzes 9-hydroxy-octadecenoic acid in comparison to 13-hydroxy-octadecenoic acid from oxidized CLs. Moreover, oxidative stress (ADP, NADPH, and Fe3+) resulted in the robust production of oxidized CLs in intact mitochondria from iPLA2γ-/- mice. In sharp contrast, oxidized CLs were readily hydrolyzed in mitochondria from wild-type mice during oxidative stress. Finally, we demonstrated that CL activates the iPLA2γ-mediated hydrolysis of arachidonic acid from phosphatidylcholine, thereby integrating the production of lipid messengers from different lipid classes in mitochondria. Collectively, these results demonstrate the integrated roles of CL and iPLA2γ in lipid second-messenger production and mitochondrial bioenergetics during oxidative stress.


Assuntos
Cardiolipinas/metabolismo , Metabolismo Energético , Fosfolipases A2 do Grupo VI/metabolismo , Mitocôndrias Cardíacas/enzimologia , Estresse Oxidativo , Transdução de Sinais , Animais , Cardiolipinas/genética , Fosfolipases A2 do Grupo VI/genética , Camundongos , Camundongos Knockout , Mitocôndrias Cardíacas/genética , Oxirredução
18.
Paediatr Int Child Health ; 36(1): 58-63, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25362964

RESUMO

OBJECTIVE: An important but neglected consequence of the AIDS pandemic that continues across sub-Saharan Africa is the phenomenon of child-headed households (CHH). This study aims to describe the challenges to health and well-being for young people living in child-headed households. METHODS: A mixed-methods research approach linked common themes using qualitative and quantitative instruments to provide a broad picture of the location and challenges of CHH in Kabira, Kyotera and Kamuganja in the Rakai District of southern Uganda. Local knowledge was used to locate CHH. RESULTS: 163 children living in 40 CHH were traced: 42·5% of the household heads were double orphans caring for younger siblings, and 43% were also caring for chronically ill or disabled grandparents who were economically unproductive and largely dependent on the eldest child for survival. It was found that those heading households were more likely not to attend school than children living at home with a parent. Their immediate needs ranged from food and shelter to health-care and education. Fear was a major theme: 38% of those interviewed reported fear of 'violence'. Children as young as 13 were responsible for navigating through complex decision-making processes from everyday basic necessities to decisions on the health care of younger siblings and grandparents. CONCLUSION: Children and young people living in CHH are a largely invisible and highly vulnerable population. Clear, officially accepted definitions of CHH are a first step in recognising this vulnerable group for whom safeguards will be necessary as social work develops in lower- and middle-income countries (LMICs). The precise numbers of CHH are unknown and further examination of this undocumented group is needed.


Assuntos
Crianças Órfãs/estatística & dados numéricos , Características da Família , Adolescente , Criança , Família , Feminino , Infecções por HIV/epidemiologia , Humanos , Masculino , Prevalência , Fatores Socioeconômicos , Inquéritos e Questionários , Uganda/epidemiologia
19.
Cell Chem Biol ; 23(10): 1217-1227, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27642067

RESUMO

Eicosanoid lipids play important roles in cellular signaling as second messengers in inflammation, immune response, vascular tone, and the CNS. Biosynthesis of eicosanoid lipids proceeds via hydrolysis of esterified arachidonic acid from phospholipids followed by oxidation of the released arachidonic acid by a variety of enzymes including cyclooxygenases (COX). Herein, we demonstrate the remarkable ability of COX-2, but not COX-1, to directly oxidize 2-arachidonoyl-lysolipids, resulting in the generation of previously unknown classes of eicosanoid-lysolipids, and provide evidence that intracellular lipases can release eicosanoids from their eicosanoid-lysolipid precursors. Importantly, genetic ablation of a phospholipase, iPLA2γ, significantly reduced the amounts of these eicosanoid-lysolipids in murine hepatic tissue and fibroblasts. Furthermore, calcium stimulation of wild-type murine lung fibroblasts produced robust increases in these eicosanoid-lysolipids, which were markedly attenuated in iPLA2γ-/- fibroblasts. Collectively, these results identify an iPLA2γ-initiated pathway generating new classes of lipid metabolites with potential signaling functions resulting from the direct COX-2 catalyzed oxidation of 2-arachidonoyl-lysolipids.


Assuntos
Ácido Araquidônico/metabolismo , Ciclo-Oxigenase 2/metabolismo , Eicosanoides/metabolismo , Lisofosfolipídeos/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Células Cultivadas , Ciclo-Oxigenase 1/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Fosfolipases A2 do Grupo VI/genética , Fosfolipases A2 do Grupo VI/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Modelos Moleculares , Oxirredução
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