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Single-Nucleotide Polymorphisms in Pig EPHX1 Gene are Associated with Pork Quality Traits
Abstract
Epoxide hydrolase 1 (EPHX1) plays an important role in both the activation and detoxification of exogenous chemicals. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the highest level of EPHX1 expression occurred in Berkshire liver, which is an organ that plays a key role in detoxification. We examined EPHX1 SNPs to analyze effect on increased expression of EPHX1 gene in Berkshire liver by total of 192 pigs of a pure Berkshire line (males = 97; females = 95). As a result, two nonsynonymous SNPs (nsSNPs) of EPHX1 were found from c.685T>G and c.776C > T, and located in 5th and 6th exons, respectively, which constitute the A/b hydrolase 1 domain of epoxide hydrolase. The nsSNP c.685T > G was significant differences in meat color, protein content, collagen content, and pH24 hr. Especially, T and G alleles of the nsSNP c.685T > G were significantly associated with CIE a*/CIE b* and protein content/pH24 hr, respectively. The nsSNP c.776C > T was significant differences in drip loss and protein content. Among meat quality traits to associate with SNPs, the protein content was only significantly associated with sex. Therefore, it is suggested that nsSNP c.685T > G in EPHX1 gene is a potential to apply as appropriate DNA markers for improvement of porcine economic traits.
... Carcasses were eviscerated and split before being placed in a chiller set at 2°-4°C for 12 h. Various meat-quality traits including carcass weight, backfat thickness, meat color (lightness, redness, and yellowness), drip loss, cooking loss, chemical composition (collagen, fat, moisture, and protein), water holding capacity, shear force, and post-mortem pH 24 h (measurement of pH after 24 h of slaughter) were measured (Cho et al., 2015b). ...
... The concentrations of moisture, crude protein, and crude fat from the LT muscles were determined according to the methods of the Association of Official Analytical Chemists (AOAC, 2006) in muscle samples taken 24 h after slaughter. Collagen content was measured as previously described (Cho et al., 2015b). The LT (6th to 13th rib) was excised and stored at 2°-4°C before it was transported to the laboratory to determine the chemical composition (Cho et al., 2015b). ...
... Collagen content was measured as previously described (Cho et al., 2015b). The LT (6th to 13th rib) was excised and stored at 2°-4°C before it was transported to the laboratory to determine the chemical composition (Cho et al., 2015b). ...
Single nucleotide polymorphisms (SNPs) are useful genetic markers that allow correlation of genetic sequences with phenotypic traits. It is shown here that HSD17B4, a bifunctional enzyme mediating dehydrogenation and anhydration during β-oxidation of long-chain fatty acids, contains a non-synonymous SNP (nsSNP) of chr2:128,825,976A>G, c.2137A>G, I690V, within the sterol carrier protein-2 domain of the HSD17B4 gene, by RNA-Seq of liver RNA. The HSD17B4 mRNA was highly expressed in the kidney and liver among various other tissues in four pig breeds, namely, Berkshire, Duroc, Landrace, and Yorkshire. The nsSNP was significantly associated with carcass weight, backfat thickness, and drip loss (P < 0.05). Furthermore, HSD17B4 may play a crucial role during the early stages of myogenesis when expression of its mRNA was significantly high. In conclusion, HSD17B4 may serve as a possible regulator of muscle development, and its identification should help to select for improved economic traits of Berkshire pigs such as carcass weight, backfat thickness, and drip loss.
... Physical property and chemical composition, including moisture content (MC), protein content (PC), fat content (FC), water holding capacity (WHC), drip loss (DL), cooking loss (CL), collagen content (CC) and meat color (L*, a*, b*), were measured according to methods employed in the previous studies (AOAC, 2000;Kristensen et al. 2001;Cho et al. 2015). ...
... Carcass and meat quality traits: Berkshire gilts reveal a tendency of higher economic efficiency than that of Berkshire barrows (Cho et al., 2015). However, an accurate assessment in accordance with Berkshire pork quality has not been classified by a statistical analysis. ...
... Therefore, the gilt appeared better than the barrow in the quality characteristics associated with taste of meat. These results have revealed in our previous study to maintain better meat quality in Berkshire gilt than the barrow (Cho et al., 2015). ...
This study was performed to analyze the difference between the meat properties classified into 5 ranges depending on Berkshire meat quality traits. Post-mortem pH24hr was positively correlated with water holding capacity, but negatively correlated with meat color, protein content, drip loss, and cooking loss. Berkshire gilt was discovered by higher population of RFN (reddish pink, firm, non-exudative), a normal meat class when compared to barrow, whereas DFD (dark, firm, and dry) was not detected and PSE (pale, soft and floppy, exudative) and RSE (reddish pink, soft and floppy, exudative) were found by a low number of individuals. It was assumed that Berkshire gilt is less susceptible to stress when compared with barrow. Therefore, from the results of this study, we suggest that Berkshire gilt is high possibility for production of good meat due to a forming ability for better meat quality than that of Berkshire barrow.
... Thus, Berkshire pork is preferred in some countries, such as Korea and Japan (Crawford et al., 2010). Therefore, previously genetic markers, such as a SNP, c.919G>A, in CBG and two SNPs, c.685T>G and c.776C>T, in EPHX1, were identified to be associated with various pork quality traits (Cho et al., 2015b). ...
... Meat quality traits, such as backfat thickness, post-mortem pH, color, WHC, postmortem temperature, Warner-Bratzler shear force, drip loss, and cooking loss, in addition to collagen, moisture, protein, fat, and ash contents, were evaluated as described previously (Cho et al., 2015b). Briefly, the longissimus dorsi muscle of Berkshire pigs (N = 416; 199 barrows and 217 gilts) was used in the experiments. ...
Cell death-inducing DFF45-like effector (CIDE) B is a member of the CIDE family of apoptosis-inducing factors. In the present study, we detected a single nucleotide polymorphism (SNP), c.414G>A, which corresponds to the synonymous SNP 414Arg, in CIDE-B in the Berkshire pigs. We also analyzed the relationships between the CIDE-B SNP and various meat quality traits. The SNP was significantly associated with post-mortem pH24h, water-holding capacity (WHC), fat content, protein content, drip loss, post-mortem temperature at 12 h (T12) and 24 h (T24) in a co-dominant model (P < 0.05). A significant association was detected between the SNP and post-mortem pH24h, fat content, protein content, drip loss, shear force, and T24 in gilts; and color parameter b*, WHC, and T24 in barrows (P < 0.05). The SNP was significantly correlated with the fat content, and CIDE-B mRNA expression was significantly upregulated during the early stage of adipogenesis, suggesting that CIDE-B may contribute towards initiation of adipogenesis (P < 0.05). Furthermore, CIDE-B mRNA was strongly expressed in the liver, kidney, large intestine, and small intestine, and weakly expressed in the stomach, lung, spleen, and white adipose tissue. These results indicate that the CIDE-B SNP is closely associated with meat quality traits and may be a useful DNA marker for improving pork quality.
... For instance, we found ZFHX4 and LPCAT1 (related to milk production, somatic cell score and conformation) [79][80][81] in YHC and MGC within the North group. We detected TNNI2, EPHX1 and ROBO1 (associated with growth, meat quality and immune response, neuron differentiation) [35,[82][83][84][85] and UBE2E1 and GNA14 (related to hematological traits and high-altitude adaptation) in PWC, LSC and ZTC within Southwest groups, respectively [86,87]. These genes can speculatively be interpreted as resulting from local adaption for mountain areas in the Southwest of China. ...
Background:
Understanding the population structure and genetic bases of well-adapted cattle breeds to local environments is one of the most essential tasks to develop appropriate genetic improvement programs.
Results:
We performed a comprehensive study to investigate the population structure, divergence and selection signatures at genome-wide level in diverse Chinese local cattle using Bovine HD SNPs array, including two breeds from North China, one breed from Northwest China, three breeds from Southwest China and two breeds from South China. Population genetic analyses revealed the genetic structures of these populations were mostly related to the geographic locations. Notably, we detected 294 and 1263 candidate regions under selection using the di and iHS approaches, respectively. A series of group-specific and breed-specific candidate genes were identified, which are involved in immune response, sexual maturation, stature related, birth and bone weight, embryonic development, coat colors and adaptation. Furthermore, haplotype diversity and network pattern for candidate genes, including LPGAT1, LCORL, PPP1R8, RXFP2 and FANCA, suggest that these genes have been under differential selection pressures in various environmental conditions.
Conclusions:
Our results shed insights into diverse selection during breed formation in Chinese local cattle. These findings may promote the application of genome-assisted breeding for well-adapted local breeds with economic and ecological importance.
... To evaluate drip loss, a slice of 2-cm thickness (weight 100± 5 g) separated from the longissimus dorsi muscle was placed into a polypropylene bag (Dongbang Co., Korea), packaged by vacuum, and then stored for 24 h at 4 o C. Drip loss was calculated by the difference in weight among samples. A slice of 3-cm thickness (weight 100±5 g) separated from the longissimus dorsi muscle for measurement of cooking loss was put into polypropylene bag (Dongbang Co., Korea), cooked for 40 min at 70 o C in a water bath, and then cooled to room temperature (Cho et al., 2015). Cooking loss was calculated by the difference in weight of the samples before and after boiling. ...
This study was performed to investigate the role of pH and temperature postmortem, and to demonstrate the importance of these factors in determining meat quality. Postmortem pH45min (pH at 45 min postmortem or initial pH) via analysis of Pearson's correlation showed high positive correlation with pH change pHc24 (pH change from pH45min to pH24h postmortem). However, postmortem pH after 24 h (pH24h or ultimate pH) had a high negative correlation with pH change, pHc24, CIE L∗, and protein content. Initial temperature postmortem (T1h) was positively associated with a change in temperature from 45 min to 24 h postmortem (Tc24) and cooking loss, but negatively correlated with water holding capacity. Temperature at 24 h postmortem (T24h) was negatively associated with Tc24. Collectively, these results indicate that higher initial pH was associated with higher pHc24, T1h, and Tc24. However, higher initial pH was associated with a reduction in carcass weight, backfat thickness, CIE a∗ and b∗, water holding capacity, collagen and fat content, drip loss, and cooking loss as well as decreased shear force. In contrast, CIE a∗ and b∗, drip loss, cooking loss, and shear force in higher ultimate pH was showed by a similar pattern to higher initial pH, whereas pHc24, carcass weight, backfat thickness, water holding capacity, fat content, moisture content, protein content, T1h, T24h, and Tc24 were exhibited by completely differential patterns (p<0.05). Therefore, we suggest that initial pH, ultimate pH, and temperatures postmortem are important factors in determining the meat quality of pork.
In mammals, Squalene epoxidase (SQLE) is an enzyme that converts squalene to 2,3-oxidosqualene, in the early stage of cholesterol generation. Here, we identified single nucleotide polymorphisms (SNPs) in the SQLE gene (c.2565 G > T) by RNA Sequencing from the liver tissue of Berkshire pigs. Furthermore, we found that homozygous GG pigs expressed more SQLE mRNA than GT heterozygous and TT homozygous pigs in longissimus dorsi tissue. Next, we showed that the SNP in the SQLE gene was associated with several meat quality traits including backfat thickness, carcass weight, meat colour (yellowness), fat composition, and water-holding capacity. Rates of myogenesis and adipogenesis induced in C2C12 cells and 3T3-L1 cells, respectively, were decreased by Sqle knockdown. Additionally, the expression of myogenic marker genes (Myog, Myod, and Myh4) and adipogenic marker genes (Pparg, Cebpa, and Adipoq) was substantially downregulated in cells transfected with Sqle siRNA. Moreover, mRNA expression levels of ROS scavengers, which affect meat quality by altering protein oxidation processes, were significantly downregulated by Sqle knockdown. Taken together, our results suggest the molecular mechanism by which SNPs in the SQLE gene can affect meat quality.
The paraoxonase (Pon) gene family contains three members: Pon1, Pon2, and Pon3. Pon3 modulates superoxide production and prevents apoptosis. The role of Pon3 has not been fully elucidated in the pig. This study is the first to investigate the association between Pon3 and meat quality in the Berkshire pig. We identified a single nucleotide polymorphism in the Pon3 gene (c.227A > G) that resulted in a change in histidine to arginine at position 76. To elucidate the role of this non-synonymous single nucleotide polymorphism in the Pon3 gene, we analysed the Pon3 genotype and meat quality traits in 434 Berkshire pigs. The results of a codominant model show that carcass weight, meat colour (lightness), cooking loss, and the Warner-Bratzler shear force were significantly associated with the Pon3 genotype. Furthermore, the 24-h post-mortem pH had the strongest relationship with the Pon3 genotype. The G allele decreased cooking loss and fat content, whereas the A allele increased the 24-h post-mortem pH and decreased backfat thickness, which contribute to meat storage life and M. longissimus dorsi depth respectively. In conclusion, the non-synonymous single nucleotide polymorphism in the Pon3 gene showed a close correlation with meat quality traits in the Berkshire pig.
Postmortem pH is a main factor influencing the meat quality in pigs. This study investigated the association of postmortem pH with single-nucleotide polymorphisms (SNPs) in the fourth member of the short-chain dehydrogenase/reductase family (DHRS4), the first member of serpin peptidase inhibitor, clade G (complement inhibitor) (SERPING1), and the apolipoprotein R precursor (APOR) genes in Berkshire pigs. The study included 437 pigs, and genotyping was conducted using the GoldenGate Assay (Illumina, San Diego, CA, USA). DHRS4, SERPING1, and APOR polymorphisms were significantly associated with pH45 or pH24 (p < 0.05). SERPING1 was also statistically significantly associated with water holding capacity (p < 0.05), which is closely associated with postmortem pH. These results suggest that SNPs in the DHRS4, SERPING1, and APOR genes have potential for use as genetic markers for the meat quality in pigs.
This study was carried out to predict the relationship between the colour and physicochemical traits in pork by using canonical correlation analysis. The variables of pork colour traits were lightness (L∗), redness (a∗) and yellowness (b∗), whereas the variables of physicochemical traits were post-mortem pH24 h, water-holding capacity (WHC), collagen content, fat content, moisture content, protein content, drip loss, cooking loss and shear force. The canonical correlation coefficient (0.819) between the first pair of canonical variates, V1 and W1, was significant (P < 0.01). According to cross loadings, drip loss, cooking loss and fat content provided the relatively high positive correlations with the variates of colour traits (V1), while pH24 h, WHC and moisture content displayed negative relationships with the variates. Otherwise, L∗and a∗strongly contribute to the variates of physicochemical traits (W1). In addition, a redundancy index of 0.256 suggests that 25.6% of the variance in V1 is explained by W1. Therefore, in order to obtain the reddish pink colour in pork that is preferred by consumers depending on the relationships between the pork quality traits, we suggest that producer leads to the proper maintenance of post-mortem pH24 h, higher WHC, lower drip loss and cooking loss in pork.
Some polymorphisms of the human CETP gene are causally and significantly associated with serum lipids levels; however, the information regarding this gene in pigs is sparse. To evaluate the effects of CETP on blood lipid traits and fat deposition in pig, porcine CETP tissue expression patterns were observed by quantitative real-time polymerase chain reaction (qPCR) first. High expression was detected in liver, spleen, gluteus medius (GM) muscle and backfat. A de novo polymorphism (AF333037:g.795C>T) in the intron 1 region of porcine CETP was identified. This polymorphism was further genotyped by direct sequencing of the PCR products of 390 Wannan Black pigs, a Chinese native breed population. Association analyses at 45 and 300 days of age revealed highly significant associations between CETP genotypes and serum lipid traits. Furthermore, this polymorphism was proved to be associated with differences in liver CETP mRNA levels: pigs at 300 days of age with the TT genotype had higher levels than did those with other genotypes (P = 0.021). Additionally, analysis at 300 days of age showed that GM CETP mRNA expression correlated positively with serum lipids levels as well as with carcass backfat thickness and intramuscular fat content in GM. These results indicate that CETP is involved in serum, adipose and muscle lipid metabolism in pigs. The mechanisms underlying such relationships and their functional implications are worthy of further research.
This study was conducted to observe the effects of crossbreeding and gender on the carcass traits and meat quality of Korean Native Black Pig (KNP) and KNP×Duroc crossbred (KNP×D). A total of 50 pigs comprising seven KNP barrows, eight KNP gilts, twenty KNP×D barrows and fifteen KNP×D gilts were used in this study. Animals were reared in the same housing condition with same feed diet for six months prior to slaughter. After an overnight chilling, the carcasses were graded, and samples of Musculus longissimus dorsi were obtained for meat quality analysis. The slaughter and carcass weights and dressing percentage of KNP×D were higher (p<0.001) than those of KNP. The slaughter and carcass weights and backfat thickness of barrows were higher (p<0.01) than those of gilts. There were no significant difference in carcass conformation and quality grade between KNP and KNP×D as well as barrow and gilt. Fat content of KNP×D was higher (p<0.001) than that of KNP. Fat content of barrow was higher (p<0.001) than that of gilt. There was interaction between crossbreeding and gender on the fat content. KNP gilt showed higher fat content than KNP barrow whereas KNP×D barrow showed higher fat content than KNP×D gilt. Lightness, redness, yellowness, chroma and hue angle values and color preference of meat of KNP×D were lower (p<0.001) than those of KNP. Redness, yellowness and chroma values of meat of barrow were lower (p<0.05) than those of gilt. It is concluded that crossbreeding KNP with Duroc increases carcass productivity and meat fat but decreases meat color values and preference. Crossbreeding of KNP with Duroc produces a better fat deposition in meat of barrows than in gilts.
CDD, the Conserved Domain Database, is part of NCBI’s Entrez query and retrieval system and is also accessible via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml. CDD provides annotation of protein sequences with the location of conserved domain footprints and functional sites inferred
from these footprints. Pre-computed annotation is available via Entrez, and interactive search services accept single protein
or nucleotide queries, as well as batch submissions of protein query sequences, utilizing RPS-BLAST to rapidly identify putative
matches. CDD incorporates several protein domain and full-length protein model collections, and maintains an active curation
effort that aims at providing fine grained classifications for major and well-characterized protein domain families, as supported
by available protein three-dimensional (3D) structure and the published literature. To this date, the majority of protein
3D structures are represented by models tracked by CDD, and CDD curators are characterizing novel families that emerge from
protein structure determination efforts.
Changes in meat quality traits are strongly associated with alterations in postmortem metabolism which depend on genetic variations, especially nonsynonymous single nucleotide variations (nsSNVs) having critical effects on protein structure and function. To selectively identify metabolism-related nsSNVs, next-generation transcriptome sequencing (RNA-Seq) was carried out using RNAs from porcine liver, which contains a diverse range of metabolic enzymes. The multiplex SNV genotyping analysis showed that various metabolism-related genes had different nsSNV alleles. Moreover, many nsSNVs were significantly associated with multiple meat quality traits. Particularly, ch7:g.22112616A>G SNV was identified to create a single amino acid change (Thr/Ala) at the 145th residue of H1.3-like protein, very close to the putative 147th threonine phosphorylation site, suggesting that the nsSNV may affect multiple meat quality traits by affecting the epigenetic regulation of postmortem metabolism-related gene expression. Besides, one nonsynonymous variation, probably generated by gene duplication, led to a stop signal in porcine testicular carbonyl reductase (PTCR), resulting in a C-terminal (E281-A288) deletion. Molecular docking and energy minimization calculations indicated that the binding affinity of wild-type PTCR to 5α-DHT, a C(21)-steroid, was superior to that of C-terminal-deleted PTCR or human carbonyl reductase, which was very consistent with experimental data, reported previously. Furthermore, P284 was identified as an important residue mediating the specific interaction between PTCR and 5α-DHT, and phylogenetic analysis showed that P284 is an evolutionarily conserved residue among animal carbonyl reductases, which suggests that the C-terminal tails of these reductases may have evolved under evolutionary pressure to increase the substrate specificity for C(21)-steroids and facilitate metabolic adaptation. Altogether, our RNA-Seq revealed that selective nsSNVs were associated with meat quality traits that could be useful for successful marker-assisted selection in pigs and also represents a useful resource to enhance understanding of protein folding, substrate specificity, and the evolution of enzymes such as carbonyl reductase.
NCBI’s Conserved Domain Database (CDD) is a resource for the annotation of protein sequences with the location of conserved
domain footprints, and functional sites inferred from these footprints. CDD includes manually curated domain models that make
use of protein 3D structure to refine domain models and provide insights into sequence/structure/function relationships. Manually
curated models are organized hierarchically if they describe domain families that are clearly related by common descent. As
CDD also imports domain family models from a variety of external sources, it is a partially redundant collection. To simplify
protein annotation, redundant models and models describing homologous families are clustered into superfamilies. By default,
domain footprints are annotated with the corresponding superfamily designation, on top of which specific annotation may indicate
high-confidence assignment of family membership. Pre-computed domain annotation is available for proteins in the Entrez/Protein
dataset, and a novel interface, Batch CD-Search, allows the computation and download of annotation for large sets of protein
queries. CDD can be accessed via http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml.
Increasing productivity is one of the main objectives in animal production. Traditional breeding methods have led to increased gains in some traits but gains are not easily attainable in traits with low heritabilities. Exploiting the genetic variations underlying desired phenotypes is the goal of today's animal producers. Such positive genetic variants must, however, be known before possible application. Consequently, candidate genes of traits of interest have been searched for possible relationships with such traits or to explain reported quantitative trait loci (QTL) for such traits. DNA variants or polymorphisms have been identified in many such genes and their relationships with production traits determined. However, only a few genes have been evaluated, given the wealth of information on reported QTL for production traits, and in most cases genes are only partially investigated. This review presents available information on DNA variants for production traits and discusses steps that are required for effective utilization of this information for successful marker-assisted selection programs.
The hydration of cis-epoxyeicosatrienoic acids to the corresponding vic-dihydroxyeicosatrienoic acids by cytosolic epoxide hydrolase demonstrates moderate regioselectivity with rates of hydration highest for the 14,15-epoxide and lower for the 11,12- and 8,9-epoxide (4.5, 1.6, and 1.5 mumol of product/mg of protein/min, respectively). Incubations of the 8,9- and 14,15-epoxides with cytosolic epoxide hydrolase show stereoselective formation of diols (7:3 and 4:1 ratio of antipodes, respectively) and concomitant chiral enrichment of the remaining unmetabolized substrate. In contrast, hydration of the 11,12-epoxide is nonenantioselective. The Km value of the enzyme for the 14(R),15(S)-epoxide is 3 microM. Incubations of the enantiomerically pure 8,9- and 14,15-epoxides with lung or liver cytosol, followed by chiral analysis of the resulting diols demonstrate selective cleavage of the oxirane ring at C9 and C15, respectively. On the other hand, cleavage of the 11,12- oxirane ring was less selective. The stereochemical preference of the cytosolic epoxide hydrolase, together with the known chiral composition of the endogenous arachidonate epoxide pools, suggests a functional role for this enzyme in the metabolism of these important compounds.
The tissue-specific pattern of mRNA expression can indicate important clues about gene function. High-density oligonucleotide arrays offer the opportunity to examine patterns of gene expression on a genome scale. Toward this end, we have designed custom arrays that interrogate the expression of the vast majority of protein-encoding human and mouse genes and have used them to profile a panel of 79 human and 61 mouse tissues. The resulting data set provides the expression patterns for thousands of predicted genes, as well as known and poorly characterized genes, from mice and humans. We have explored this data set for global trends in gene expression, evaluated commonly used lines of evidence in gene prediction methodologies, and investigated patterns indicative of chromosomal organization of transcription. We describe hundreds of regions of correlated transcription and show that some are subject to both tissue and parental allele-specific expression, suggesting a link between spatial expression and imprinting.
Thousands of quantitative trait loci (QTL) have been identified for a wide range of economically important phenotypes in pigs. Recently, QTL analyses have begun to use high-density single nucleotide polymorphism (SNP) panels and applications have extended beyond experimental intercrosses to outbred populations by exploiting long-range linkage disequilibrium that results in higher resolution QTL mapping. Relevant phenotypes generally fall under categories of growth and body composition, carcass and meat quality, reproduction, and disease resistance. A few expression QTL (eQTL) studies have been performed that integrate transcriptional profiles with genotype data by considering expression levels as response variables in QTL analyses for identifying genes controlling important trait phenotypes. Rapidly evolving genomics technologies, including RNAseq, provide tremendous opportunities for QTL and eQTL discovery. In this review, we discuss recent progress in pig QTL and eQTL discovery, including approaches for allele-specific expression, and implications of these discoveries for pig breeding and genetics.
The water-holding capacity (WHC) of pork decreases post-mortem but has been shown to increase during subsequent ageing. In order to test a hypothesis that water-holding capacity increases during ageing due to degradation of the cytoskeleton, WHC was followed 10 days post-mortem and related to the extent of proteolysis of cytoskeletal proteins. A fast method for measuring WHC in small meat samples was developed by the use of centrifugation. The WHC of fresh pork decreases in the first part of post-mortem storage after which it increases to the level of 1 day PM. No changes in total water content of the meat were observed which could explain changes in WHC during ageing. Vinculin and desmin degrade gradually during ageing while talin degrades rapidly. These observations are consistent with the hypothesis that degradation of the cytoskeleton slowly removes the linkage between lateral shrinkage of myofibrils and shrinkage of entire muscle fibres, so removing the force that causes flow into the extracellular space. Inflow of previously expelled water is then possible, so increasing WHC as observed in later periods of storage.
Subcutaneous fat tissues from an indigenous fat-type breed and an intensively-lean selected breed were studied in juvenile pigs. Combining DIGE with bioinformatics and target analyses of key genes, enzymes or terminal routes, this study identifies metabolic and homeostatic processes, response to organic substances, and acute-phase responses as the main pathways whose proteins were regulated in association with adiposity. Breed-related differences in abundance and activities of malic enzyme and glucose-6-phosphate dehydrogenase NADPH-supplying enzymes suggested up-regulation of the lipogenic pathway to dispose for a greater adiposity. Over-abundance in the lipolytic protein carboxylesterase-1 was revealed in fat-type piglets. A panel of pro- and anti-inflammatory proteins such as serpins, had an altered abundance in the fat-type piglets, suggesting adverse consequences of fat accumulation even in early post-weaning stages. Propensity to low-grade inflammation in fat pigs was reinforced by the up-regulation of genes encoding pro-inflammatory cytokines IL6 and TNF-α in these piglets. Differential abundance in annexin-A5 and pericentrin suggested a positive regulation of cell apoptosis in lean piglets. Our results are relevant in the context of data linking the accretion of body lipids to the physiology and pathology of adipose tissue in models other than rodents for a better control of human health and nutrition.
Obesity is an increasingly important public health issue reaching epidemic proportions. Visceral obesity has been defined as an important element of the metabolic syndrome and expansion of the visceral fat mass has been shown to contribute to the development of insulin resistance and cardiovascular disease. To identify novel contributors to cardiovascular and metabolic abnormalities in obesity, we analyzed the adipose proteome and identified soluble epoxide hydrolase (sEH) in the epididymal fat pad from C57BL/6J mice that received either a regular diet or a "western diet." sEH was synthesized in adipocytes and expression levels increased upon differentiation of 3T3-L1 preadipocytes. Although normalized sEH mRNA and protein levels did not differ in the fat pads from mice receiving a regular or a "western diet," total adipose sEH activity was higher in the obese mice, even after normalization for body weight. Furthermore, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increased the expression of sEH in mature 3T3-L1 adipocytes in vitro and in adipose tissue in vivo. Considering the established role for sEH in inflammation, cardiovascular diseases, and lipid metabolism, and the suggested involvement of sEH in the development of type 2 diabetes, our study has identified adipose sEH as a potential novel therapeutic target that might affect the development of metabolic and cardiovascular abnormalities in obesity.
Using trans-stilbene oxide and styrene oxide as substrates, epoxide hydrolase activities were measured in cytosolic and microsomal fractions from liver, kidney, heart, lung and testis of male DBA/2 mice. The activities towards these two substrates are remarkably organ specific: trans-stilbene oxide was most effectively hydrolyzed in subcellular fractions from liver, kidney and heart, whereas styrene oxide was predominantly hydrolyzed in those from liver, lung and testis. Immunoblotting experiments were performed with two polyclonal antibodies isolated from goat antisera. Using an anti-mouse liver cytosolic epoxide hydrolase antibody, the corresponding antigen protein was predominantly detected in both cytosolic and microsomal fractions from liver, kidney and heart. An anti-rat liver microsomal epoxide hydrolase antibody proved to be cross-reactive with the mouse enzyme and stained SDS-gels run with microsomal fractions from liver, lung and testis. The anti-mouse liver cytosolic epoxide hydrolase antibody precipitated cytosolic epoxide hydrolase activities from liver, kidney and heart cytosolic fractions. Dietary exposure to the hypolipidemic agent nafenopin (2000 ppm/10 days) caused an induction of trans-stilbene oxide hydrolase and styrene oxide hydrolase activities in cytosolic and microsomal liver fractions whereas, in the other organs, the same activities were unaffected by this treatment. This finding was in accordance with the increased amounts of antigen protein as detected with the antibodies in liver fractions from treated animals. The anti-mouse liver cytosolic epoxide hydrolase antibody was found to precipitate the whole trans-stilbene oxide hydrolase activity also from liver cytosol of nafenopin-treated mice, which indicates the presence of a single cytosolic epoxide hydrolase following induction.
Epoxyeicosatrienoic acids, formed during the cytochrome P-450-catalyzed oxidation of arachidonic acid, react with a liver cytosolic epoxide hydrolase to form vicinal diols of eicosatrienoic acid. The role of this cytosolic enzyme, rather than a microsomal bound type, explains previous results illustrating the ability to accumulate epoxides during the in vitro aerobic steady state of oxidative metabolism of arachidonic acid by liver microsomes. The inability of the 5,6-epoxyeicosatrienoic acid to serve as a suitable substrate for this enzyme is discussed in light of recent studies concerning possible unique physiological functions for this metabolite.
Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in Km and Vmax. In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37 degrees C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.
Mycobacterium tuberculosis (Mtb), the intracellular pathogen that infects macrophages primarily, is the causative agent of the infectious disease tuberculosis in humans. The Mtb genome encodes at least six epoxide hydrolases (EHs A to F). EHs convert epoxides to trans-dihydrodiols and have roles in drug metabolism as well as in the processing of signaling molecules. Herein, we report the crystal structures of unbound Mtb EHB and Mtb EHB bound to a potent, low-nanomolar (IC(50) approximately 19 nM) urea-based inhibitor at 2.1 and 2.4 A resolution, respectively. The enzyme is a homodimer; each monomer adopts the classical alpha/beta hydrolase fold that composes the catalytic domain; there is a cap domain that regulates access to the active site. The catalytic triad, comprising Asp104, His333 and Asp302, protrudes from the catalytic domain into the substrate binding cavity between the two domains. The urea portion of the inhibitor is bound in the catalytic cavity, mimicking, in part, the substrate binding; the two urea nitrogen atoms donate hydrogen bonds to the nucleophilic carboxylate of Asp104, and the carbonyl oxygen of the urea moiety receives hydrogen bonds from the phenolic oxygen atoms of Tyr164 and Tyr272. The phenolic oxygen groups of these two residues provide electrophilic assistance during the epoxide hydrolytic cleavage. Upon inhibitor binding, the binding-site residues undergo subtle structural rearrangement. In particular, the side chain of Ile137 exhibits a rotation of around 120 degrees about its C(alpha)-C(beta) bond in order to accommodate the inhibitor. These findings have not only shed light on the enzyme mechanism but also have opened a path for the development of potent inhibitors with good pharmacokinetic profiles against all Mtb EHs of the alpha/beta type.
CDD-a conserved domain database for the functional annotation of proteins
- A Marchler-Bauer
- S Lu
- Jb Anderson
- F Chitsaz
- Mk Derbyshire
- C Deweese-Scott
- Jh Fong
- Ly Geer
- Rc Geer
- Nr Gonzales
- M Gwadz
- Di Hurwitz
- Jd Jackson
- Z Ke
- Cj Lanczycki
- F Lu
- Gh Marchler
- M Mullokandov
- Mv Omelchenko
- Cl Robertson