Deficiency of cardiac Acyl-CoA synthetase-1 induces diastolic dysfunction,but pathologic hypertrophy is reversed by rapamycin

In mice with temporally-induced cardiac-specific deficiency of acyl-CoA synthetase-1 (Acsl1^H −/−), the heart is unable to oxidize long-chain fatty acids and relies primarily on glucose for energy. These metabolic changes result in the development of both a spontaneous cardiac hypertrophy and increased phosphorylated S6 kinase (S6K), a substrate of the mechanistic target of rapamycin, mTOR. Doppler echocardiography revealed evidence of significant diastolic dysfunction, indicated by a reduced E/A ratio and increased mean performance index, although the deceleration time and the expression of sarco/endoplasmic reticulum calcium ATPase and phospholamban showed no difference between genotypes. To determine the role of mTOR in the development of cardiac hypertrophy, we treated Acsl1^H −/− mice with rapamycin. Six to eight week old Acsl1^H −/− mice and their littermate controls were given i.p. tamoxifen to eliminate cardiac Acsl1, then concomitantly treated for 10 weeks with i.p. rapamycin or vehicle alone. Rapamycin completely blocked the enhanced ventricular S6K phosphorylation and cardiac hypertrophy and attenuated the expression of hypertrophy-associated fetal genes, including α-skeletal actin and B-type natriuretic peptide. mTOR activation of the related Acsl3 gene, usually associated with pathologic hypertrophy, was also attenuated in the Acsl1^H −/− hearts, indicating that alternative pathways of fatty acid activation did not compensate for the loss of Acsl1. Compared to controls, Acsl1^H −/− hearts exhibited an 8-fold higher uptake of 2-deoxy[1-¹⁴C]glucose and a 35% lower uptake of the fatty acid analog 2-bromo[1-¹⁴C]palmitate. These data indicate that Acsl1-deficiency causes diastolic dysfunction and that mTOR activation is linked to the development of cardiac hypertrophy in Acsl1^H −/− mice.     

Molecular characterization and tissue-specific expression of the acetyl-CoA carboxylase α gene from Grass carp, Ctenopharyngodon idella

Acetyl-CoA carboxylase α (ACC1), the major regulatory enzyme of fatty acid biosynthesis, catalyzes the conversion of acetyl-CoA to malonyl-CoA. The full-length cDNA coding ACC1 isoform was cloned from liver of grass carp. The cDNA obtained was 7515 bp with a 7173 bp open reading frame encoding 2389 amino acids. The ACC1 protein has a calculated molecular weight of 269.2 kDa and isoelectric point of 6.23. Tissue distribution of ACC1 mRNA in brain, mesenteric adipose, spleen, white muscle and liver of grass carp was analyzed by real-time PCR method using β-actin as an internal control for cDNA normalization. The results showed that the expressions of ACC1 mRNA were detected in all examined tissues. Relative expression profile of ACC1 mRNA in liver normalized with β-actin level was 15, 92, 135 and 165-fold compared with the level in brain, white muscle, mesenteric adipose and spleen, respectively. In addition, we present evidence for the presence of two isoforms of ACC1 (265.7 kDa and 267.2 kDa) in grass carp liver that differ from the 269.2 kDa ACC1 by the absence of 34 and 15 amino acids. In conclusion, the liver is one of the main ACC1 producing tissues in grass carp and ACC1 gene was highly homologous to that of mammals.     

Molecular cloning and tissue distribution of the phosphotyrosine interaction domain containing 1 (PID1) gene in Tianfu goat

Phosphotyrosine interaction domain containing 1 (PID1) is an important mediator in the development of obesity-related insulin resistance in humans and animals. For a better understanding of the structure and function of the PID1 gene and to study its effect in caprine, the cDNA of the PID1 gene from the abdominal muscle of Tianfu goat was cloned and sequenced. The structure of PID1 was analyzed using bioinformatics tools. The results showed that the full sequence of the caprine PID1 cDNA was 896 bp long and contained a 654 bp long coding region that encoded a 217 amino acid sequence. Fifteen phosphorylation sites were predicted in the translated PID1 protein. The protein had a phosphotyrosine-binding domain between Arg⁵³ and Ile¹⁹⁹. A phylogenic tree based on the PID1 proteins from other species revealed that the caprine protein was closely related to cattle PID1. Fluorescence quantitative PCR analyses revealed that PID1 was expressed in the heart, liver, spleen, lung, kidney, leg muscle, abdominal muscle and longissimus dorsi muscle of goats. In particular, high expression levels of PID1 were detected in liver and abdominal muscle, and low expression levels were seen in lung. Furthermore, the PID1 mRNA expression levels in the longissimus dorsi muscles increased gradually with the age of the goats (P < 0.05). Western blotting results detected the PID1 protein in six of the tissues in which PID1 was shown to be expressed; the two exceptions were liver and spleen.     

Cloning, characterization, and expression analysis of Toll-like receptor-7 cDNA from common carp, Cyprinus carpio L.

The Toll-like receptor 7 (TLR7) is activated by single strand RNA and RNA-like compounds (imidazoquinoline), and it induces interferon production. We identified and described carp TLR7 cDNA and its mRNA expression. The full-length cDNA of carp TLR7 gene is 3427 bp, encoding 1049 amino acids (AB553573). The similarities of carp TLR7 with zebrafish, rainbow trout, fugu, and human TLR7 were 89.6, 83.4, 80.6 and 74.6%, respectively, at the amino acid sequence level. Furthermore, the expression of TLR7 mRNA was investigated in normal tissues of carp by semi-quantitative RT-PCR analysis. Carp TLR7 expression was exhibited in healthy tissues (kidney, brain, spleen, skin, intestine, muscle, liver, gills and heart) and though the expression level in each tissue varied among healthy fish. Carp TLR7 expression was significantly increased in head kidney stimulated with TLR7 agonist, imiquimod, at 8, 24 and 48 h in vitro when compared to expression in the control group. Moreover, carp head kidney leukocytes produced elevated levels of pro-inflammatory and type 1 interferon cytokine mRNA in response to imiquimod stimulation.     

Molecular cloning and characterization of the β-catenin gene from fine-wool sheep

β-Catenin is an evolutionarily conserved molecule that functions as a crucial effector in both cell-to-cell adhesion and Wnt signaling. To gain a better understanding of its role in the development of hair follicles, we cloned the cDNA sequence of the β-catenin gene from the skin of Aohan fine-wool sheep and performed a variety of bioinformatics analyses. We obtained the full-length sequence, which was 4573-bp long and contained a 2346-bp open reading frame encoding a protein of 781 amino acids. The protein had a predicted molecular weight of 85.4 kDa and a theoretical isoelectric point of 5.57. Domain architecture analysis of the β-catenin protein revealed an armadillo repeat region, which is a common feature of β-catenin in other species. The ovine β-catenin gene shares 97.91%, 94.25%, 94.59%, 83.89%, and 89.39% sequence identity with its homologs in Bos taurus, Homo sapiens, Sus scrofa, Gallus gallus, and Mus musculus, respectively, while the amino acid sequence is more than 99% identical with each of these species. The expression of β-catenin mRNA was detected in the heart, liver, spleen, lung, kidney, skin, muscle, and adipose tissue. Expression levels were maximal in the lung and minimal in the muscle, and the difference in expression in these tissues was significant (P < 0.01). Western blot analysis revealed the presence of the β-catenin protein in all tissues examined; expression was lowest in the skin and adipose tissues.     

Cloning,characterization, hypoxia and heat shock response of hypoxia inducible factor-1 (HIF-1) from the small abalone Haliotis diversicolor

In this study, hypoxia inducible factor-1α (HIF-1α) and hypoxia inducible factor-1β (HIF-1β) from small abalone Haliotis diversicolor were cloned. The cDNA of H. diversicolor HIF-1α (HdHIF-1α) is 2833 bp encoding a protein of 711aa and H. diversicolor HIF-1β (HdHIF-1β) is 1919 bp encoding a protein of 590aa. Similar to other species' HIF-1, HdHIF-1 has one basic helix–loop–helix (bHLH) domain and two Per-Arnt-Sim (PAS) domains, and HdHIF-1α has a oxygen-dependent degradation domain (ODDD) with two proline hydroxylation motifs and a C-terminal transactivation domain (C-TAD) with an asparagine hydroxylation motif. Under normoxic conditions, HdHIF-1α and HdHIF-1β mRNAs were constitutively present in all examined tissues. Under hypoxia (2.0 mg/L DO at 25 °C) stress, HdHIF-1α expression was up-regulated in gills at 4 h, 24 h and 96 h, and in hemocytes at 24 h and 96 h, while HdHIF-1β remained relatively constant. Under thermal stress (31 °C), HdHIF-1α expression was significantly increased in gills at 4 h, and hemocytes at 0 h and 4 h, while HdHIF-1β expression still remained relatively constant. These results suggested that HIF-1α may play an important role in adaption to poor environment in H. diversicolor.     

Characterization,expression and localization of valosin-containing protein in ovaries of the giant tiger shrimp Penaeus monodon

Valosin-containing protein (VCP), a member of the ATPase-associated with diverse cellular activity (AAA) family, was identified from the giant tiger shrimp (Penaeus monodon). The full-length cDNA of the PmVCP mRNA consisted of 2724 bp containing an ORF of 2367 bp corresponding to a deduced polypeptide of 788 amino acids. The deduced PmVCP protein contained two putative Cdc48 domains (positions 17–103, E-value = 2.00e− 36 and 120–186, E-value = 3.60e− 11) and two putative AAA domains (positions 232–368, E-value = 3.67e− 24 and 505–644, E-value = 3.73e− 25). PmVCP mRNA expression in ovaries was greater than that in testes in both juveniles and broodstock. PmVCP was significantly up-regulated in stages II and IV ovaries in intact wild broodstock (P < 0.05) but it was not differentially expressed during ovarian development in eyestalk-ablated broodstock (P > 0.05). The expression level of PmVCP mRNA in ovaries of 14-month-old shrimp was not affected by progesterone injection (0.1 μg/g body weight, P > 0.05). In contrast, exogenous 5-HT administration (50 μg/g body weight) resulted in an increase of PmVCP mRNA in ovaries of 18-month-old shrimp at 6 and 24 h post-injection (hpi) (P < 0.05). The rPmCdc48-VCP protein and its polyclonal antibody were successfully produced. Cellular localization revealed that PmVCP was localized in the ooplasm of previtellogenic oocytes. Subsequently, it was translocated into the germinal vesicle of vitellogenic oocytes. Interestingly, PmVCP was found in nucleo-cytoplasmic compartments, in the cytoskeletal architecture and in the plasma membrane of mature oocytes in both intact and eyestalk-ablated broodstock.     

Inhibition of mTORC1 induces loss of E-cadherin through AKT/GSK-3β signaling-mediated upregulation of E-cadherin repressor complexes in non-small cell lung cancer cells

Background

mTOR, which can form mTOR Complex 1 (mTORC1) or mTOR Complex 2 (mTORC2) depending on its binding partners, is frequently deregulated in the pulmonary neoplastic conditions and interstitial lung diseases of the patients treated with rapalogs. In this study, we investigated the relationship between mTOR signaling and epithelial mesenchymal transition (EMT) by dissecting mTOR pathways.

Methods

Components of mTOR signaling pathway were silenced by shRNA in a panel of non-small cell lung cancer cell lines and protein expression of epithelial and mesenchymal markers were evaluated by immunoblotting and immunocytochemistry. mRNA level of the E-cadherin repressor complexes were evaluated by qRT-PCR.

Results

IGF-1 treatment decreased expression of the E-cadherin and rapamycin increased its expression, suggesting hyperactivation of mTOR signaling relates to the loss of E-cadherin. Genetic ablation of rapamycin-insensitive companion of mTOR (Rictor), a component of mTORC2, did not influence E-cadherin expression, whereas genetic ablation of regulatory-associated protein of mTOR (Raptor), a component of mTORC1, led to a decrease in E-cadherin expression at the mRNA level. Increased phosphorylation of AKT at Ser473 and GSK-3β at Ser9 were observed in the Raptor-silenced NSCLC cells. Of the E-cadherin repressor complexes tested, Snail, Zeb2, and Twist1 mRNAs were elevated in raptor-silenced A549 cells, and Zeb2 and Twist1 mRNAs were elevated in Raptor-silenced H2009 cells. These findings were recapitulated by treatment with the GSK-3β inhibitor, LiCl. Raptor knockdown A549 cells showed increased expression of N-cadherin and vimentin with mesenchymal phenotypic changes.

Conclusions

In conclusion, selective inhibition of mTORC1 leads to hyperactivation of the AKT/GSK-3β pathway, inducing E-cadherin repressor complexes and EMT. These findings imply the existence of a feedback inhibition loop of mTORC1 onto mTORC2 that plays a role in the homeostasis of E-cadherin expression and EMT, requiring caution in the clinical use of rapalog and selective mTORC1 inhibitors.     

Calcium–calmodulin dependent protein kinase I from Macrobrachium nipponense: cDNA cloning and involvement in molting

Calcium–calmodulin dependent protein kinase I is a component of a calmodulin-dependent protein kinase cascade and involved in many physiological processes. The full-length cDNA of calcium–calmodulin dependent protein kinase I (MnCaMKI) was cloned from the freshwater prawn Macrobrachium nipponense and its expression pattern during the molt cycle and after eyestalk ablation is described. The full-length cDNA of MnCaMKI is 3262 bp in length and has an open reading frame (ORF) of 1038 bp, encoding a 345 amino acid protein. The expression of MnCaMKI in three examined tissues was upregulated in the premolt stage of the molt cycle. Its expression was induced after eyestalk ablation (ESA): the highest expression level was reached 1 day after ESA in hepatopancreas, and 3 days after ESA in muscle. By dsRNA-mediated RNA interference assay, expression of MnCaMKI and ecydone receptor gene (MnEcR) was significantly decreased in prawns treated by injection of dsMnCaMKI, while expression of these two genes was also significantly decreased in prawns treated by injection of dsMnEcR, demonstrating a close correlation between the expression of these two genes. These results suggest that CaMKI in M. nipponense is involved in molting.     

Molecular characterization,transcriptional regulation and association analysis with carcass traits of porcine TCAP gene

TCAP (also known as titin-cap or telethonin) is one of the titin interacting Z-disk proteins involved in the regulation and development of normal sarcomeric structure. In this study, we cloned the cDNA and promoter sequences of porcine TCAP gene, which contained a 504 bp full-length coding region. Quantitative real-time PCR (qRT-PCR) analyses showed that porcine TCAP was highly expressed in the skeletal muscle, heart, and kidney. During postnatal muscle development, TCAP expression was down-regulated from 30 days to 120 days in Large White and Meishan pigs. One single nucleotide polymorphism c.334G>A in exon 2 of the TCAP gene was identified and detected by allele-specific primer-polymerase chain reaction (ASP-PCR). Association analysis revealed that the polymorphism had significant associations (P < 0.05 and P < 0.01) with some carcass traits. Analysis of the porcine TCAP promoter in different cell lines demonstrated that it is a muscle-specific promoter. In addition, we found that the porcine TCAP promoter can be activated by MyoD, MyoG and MEF2 in myotubes, which indicated that TCAP may play a role in the regulation of porcine skeletal muscle development. These findings provide useful information for the further investigation of the function of TCAP in porcine skeletal muscle.     

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5 mg/kg) or vehicle. After 10 weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P < 0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P < 0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.     

Cloning,characterization, and expression analysis of acyl–acyl carrier protein (ACP)-thioesterase B from seeds of Chinese Spicehush (Lindera communis)

Acyl–acyl carrier protein (ACP) thioesterases (TE EC 3.1.2.14) are fatty acid biosynthesis key enzymes that determine fatty acid carbon chain length in most plant tissues. A full-length cDNA corresponding to one of the fatty acyl–ACP thioesterase (Fat) genes, designated LcFatB, was isolated from developing Lindera communis seeds using PCR and RACE with degenerate primers based on conserved sequences of multiple TE gene sequences obtained from GenBank. The 1788 bp cDNA had an open reading frame (ORF) of 1260 bp encoding a protein of 419 amino acids. The deduced amino acid sequence showed 61–73% identity to proteins in the FatB class of plant thioesterases. Real-time quantitative PCR analysis revealed that LcFatB was expressed in all tissues of L. communis, with the highest expression in the developing seeds 75 days after flowering. Recombinant pET-MLcFatB was constructed using the pET-30a vector and transformed into Escherichia coli BL21(DE3)△ FadE, a strain that deleted the acyl-CoA dehydrogenase (FadE). SDS-PAGE analysis of proteins isolated from pET-MLcFatB E. coli cells after induction with IPTG revealed a protein band at ~ 40.5 kDa, corresponding to the predicted size of LcFatB mature protein. The decanoic acid and lauric acid contents of the pET-MLcFatB transformant were increased significantly. These findings suggest that an LcFatB gene from a non-traditional oil-seed tree could be used to function as a saturated acyl–ACP thioesterase and could potentially be used to modify the fatty acid composition of seed oil from L. communis or other species through transgenic approaches.     

SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein

In a recent proteomic study of lysosomal proteins [10], we identified SID1 transmembrane family, member 2 (Sidt2) as a novel lysosomal membrane protein candidate. The Sidt2 gene encodes an 832-amino acid residues protein with a calculated molecular mass of 94.5 kDa. Bioinformatic analysis showed that Sidt2 is a multipass transmembrane protein that contains 10 putative N-glycosylation sites (NxS/T) and two potential tyrosine-based sorting signals (YGSF and YDTL). Using specific anti-Sidt2 antibody and lysosomal markers, the lysosomal localization of Sidt2 was determined by immunofluorescence. Furthermore, using subcellular fractionation techniques, we demonstrated that Sidt2 is a lysosomal integral membrane protein. Endogenous Sidt2 was detected in multiple tissues of mouse and rat with approximately 120-130 kDa molecular weights due to extensive glycosylation. After digestion with PNGase F, the apparent molecular mass of Sidt2 decreased to the predicted value of 95 kDa. In rats, Sidt2 was highly expressed in the liver, brain, and kidney, whereas no or little expression was found in the skeletal muscles, heart, and other tissues. In summary, Sidt2 is a highly glycosylated lysosomal integral membrane protein that shows tissue-specific expression.     

Characterization of myosin light chain gene up-regulated in the large yellow croaker immunity by interaction with RanGTPase

RanGTPases are highly conserved in eukaryotes from yeast to human and have been implicated in many aspects of nuclear structure and function. In our previous study, it was revealed that the RanGTPase was up-regulated in large yellow croaker challenged by pathogen. However, the mechanism of RanGTPase in immunity remains unclear. In this investigation, on the basis of protein interaction, it was found that RanGTPase interacted with myosin light chain (designated as LycMLC), a crucial protein in the process of phagocytosis. Furthermore, it was found and characterized in this marine fish for the first time. The full-length cDNA of LycMLC was 771 bp, including a 5′-terminal untranslated region (UTR) of 36 bp, 3′-terminal UTR of 279 bp and an open reading frame (ORF) of 456 bp encoding a polypeptide of 151 amino acids. RT-PCR analysis indicated that LycMLC gene was constitutively expressed in the 9 tissues examined, including kidney, liver, gill, muscle, spleen, skin, heart, intestine and blood. The result of quantitative real-time PCR analysis revealed the highest expression in muscle and the weakest expression in skin. Time course analysis showed that LycMLC expression was obviously up-regulated in blood after immunization with either poly I:C or formalin-inactive Gram-negative bacteria Vibrio parahaemolyticus. It indicated that the highest expression was 4.5 times (at 24 h) as much as that in the control (P < 0.05) challenged by poly I:C and 5.0 times (at 24 h) challenged by bacteria. These results suggested that LycMLC might play an important role in large yellow croaker defense against the pathogen infection. Therefore our study revealed a novel pathway concerning immunity of RanGTPase by the direct interaction with the cytoskeleton protein, which would help to better understand the molecular events in immune response against pathogen infection in fish.     

Molecular cloning,characterization and expression of myoglobin in Tibetan antelope (Pantholops hodgsonii), a species with hypoxic tolerance

The Tibetan antelope (Pantholops hodgsonii) is a hypoxia-tolerant species that lives at an altitude of 4000–5000 m above sea level on the Qinghai–Tibetan plateau. Myoglobin is an oxygen-binding cytoplasmic hemoprotein that is abundantly expressed in oxidative skeletal and cardiac myocytes. Numerous studies have implicated that hypoxia regulates myoglobin expression to allow adaptation to conditions of hypoxic stress. Few studies have yet looked at the effect of myoglobin on the adaptation to severe environmental stress on TA. To investigate how the Tibetan antelope (TA) has adapted to a high altitude environment at the molecular level, we cloned and analyzed the myoglobin gene from TA, compared the expression of myoglobin mRNA and protein in cardiac and skeletal muscle between TA and low altitude sheep. The results indicated that the full-length myoglobin cDNA is composed of 1154 bp with a 111 bp 5′ untranslated region (UTR), a 578 bp 3′ UTR and a 465 bp open reading frame (ORF) encoding a polypeptide of 154 amino acid residues with a predicted molecular weight of 17.05 kD. The TA myoglobin cDNA sequence and the deduced amino acid sequence were highly homologous with that of other species. When comparing the myoglobin sequence from TA with the Ovis aries myoglobin sequence, variations were observed at codons 21 (GGT → GAT) and 78 (GAA → AAG), and these variations lead to changes in the corresponding amino acids, i.e., Gly → Asp and Glu → Lys, respectively. But these amino acid substitutions are unlikely to effect the ability of binding oxygen because their location is less important, which is revealed by the secondary structure and 3D structure of TA myoglobin elaborated by homology modeling. However, the results of myoglobin expression in cardiac and skeletal muscles showed that they were both significantly higher than that in plain sheep not only in mRNA but also protein level. We speculated that the higher expression of myoglobin in TA cardiac and skeletal muscles improves their ability to obtain and store oxygen under hypoxic conditions. This study indicated that TA didn't improve the ability of carrying oxygen by changing the molecular structure of myoglobin, but through increasing the expression of myoglobin in cardiac and skeletal muscles.     

High-frequency electrically stimulated skeletal muscle contractions increase p70 phosphorylation independent of known IGF-I sensitive signaling pathways

Insulin-like growth factor (IGF-I) is hypothesized to be a critical upstream regulator of mammalian target of rapamycin (mTOR)-regulated protein synthesis with muscle contraction. We utilized a mouse model that expresses a skeletal muscle specific dominant-negative IGF-I receptor to investigate the role of IGF-I signaling of protein synthesis in response to unilateral lengthening contractions (10 sets, 6 repetitions, 100 Hz) at 0 and 3 h following the stimulus. Our results indicate that one session of high frequency muscle contractions can activate mTOR signaling independent of signaling components directly downstream of the receptor.