Molecular characterization of argininosuccinate synthase and argininosuccinate lyase from the liver of the African lungfish Protopterus annectens,and their mRNA expression levels in the liver,kidney, brain and skeletal muscle during aestivation

Argininosuccinate synthase (Ass) and argininosuccinate lyase (Asl) are involved in arginine synthesis for various purposes. The complete cDNA coding sequences of ass and asl from the liver of Protopterus annectens consisted of 1,296 and 1,398 bp, respectively. Phylogenetic analyses revealed that the deduced Ass and Asl of P. annectens had close relationship with that of the cartilaginous fish Callorhinchus milii. Besides being strongly expressed in the liver, ass and asl expression were detectable in many tissues/organs. In the liver, mRNA expression levels of ass and asl increased significantly during the induction phase of aestivation, probably to increase arginine production to support increased urea synthesis. The increases in ass and asl mRNA expression levels during the prolonged maintenance phase and early arousal phase of aestivation could reflect increased demand on arginine for nitric oxide (NO) production in the liver. In the kidney, there was a significant decrease in ass mRNA expression level after 6 months of aestivation, indicating possible decreases in the synthesis and supply of arginine to other tissues/organs. In the brain, changes in ass and asl mRNA expression levels during the three phases of aestivation could be related to the supply of arginine for NO synthesis in response to conditions that resemble ischaemia and ischaemia–reperfusion during the maintenance and arousal phase of aestivation, respectively. The decrease in ass mRNA expression level, accompanied with decreases in the concentrations of arginine and NO, in the skeletal muscle of aestivating P. annectens might ameliorate the potential of disuse muscle atrophy.     

Expression of Transthyretin during bovine myogenic satellite cell differentiation

Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T₄) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T₄ decreased significantly following TTR knockdown, indicating the critical role of TTR in T₄ transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T₄ and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.     

Interaction of diet and the masou salmon Δ5-desaturase transgene on Δ6-desaturase and stearoyl-CoA desaturase gene expression and N-3 fatty acid level in common carp (Cyprinus carpio)

The masou salmon Δ5-desaturase-like gene (D5D) driven by the common carp β-actin promoter was transferred into common carp (Cyprinus carpio) that were fed two diets. For P₁ transgenic fish fed a commercial diet, Δ6-desaturase-like gene (D6D) and stearoyl-CoA desaturase (SCD) mRNA levels in muscle were up-regulated (P < 0.05) 12.7- and 17.9-fold, respectively, and the D6D mRNA level in the gonad of transgenic fish was up-regulated 6.9-fold (P < 0.05) compared to that of non-transgenic fish. In contrast, D6D and SCD mRNA levels in transgenic fish were dramatically down-regulated (P < 0.05), 50.2- and 16.7-fold in brain, and 5.4- and 2.4-fold in liver, respectively, in comparison with those of non-transgenic fish. When fed a specially formulated diet, D6D and SCD mRNA levels in muscle of transgenic fish were up-regulated (P < 0.05) 41.5- and 8.9-fold, respectively, and in liver 6.0- and 3.3-fold, respectively, compared to those of non-transgenic fish. In contrast, D6D and SCD mRNA levels in the gonad of transgenic fish were down-regulated (P < 0.05) 5.5- and 12.4-fold, respectively, and D6D and SCD mRNA levels in the brain were down-regulated 14.9- and 1.4-fold (P < 0.05), respectively, compared to those of non-transgenic fish. The transgenic common carp fed the commercial diet had 1.07-fold EPA, 1.12-fold DPA, 1.07-fold DHA, and 1.07-fold higher observed total omega-3 fatty acid levels than non-transgenic common carp. Although these differences were not statistically different (P > 0.05), there were significantly (P < 0.10) higher omega-3 fatty acid levels when considering the differences for all of the individual omega-3 fatty acids. The genotype × diet interactions observed indicated that the potential of desaturase transgenesis cannot be realized without using a well-designed diet with the needed amount of substrates.     

Possible Correlation of Selenoprotein W with Inflammation Factors in Chicken Skeletal Muscles

The aim of the present study was to investigate the possible correlation of selenoprotein W (SelW) with inflammatory injury induced by dietary selenium (Se) deficiency in chicken. One-day-old male chickens were fed either a commercial diet or a Se-deficient diet for 55 days. Then, the expression levels of SelW messenger RNA (mRNA) and inflammation-related genes (NF-κB, TNF-α, iNOS, COX-2, and PTGES) in chicken skeletal muscles (wing muscle, pectoral muscle, and thigh muscle) were determined at 15, 25, 35, 45, and 55 days old, respectively. In addition, the correlation between SelW mRNA expression and inflammation-related genes were assessed. The results showed that dietary Se deficiency reduced the mRNA expression of SelW in chicken wing, pectorals, and thigh muscles. In contrast, Se deficiency increased the mRNA expression levels of inflammation-related genes in chicken skeletal muscle tissues at different time points. The Pearson’s correlation coefficients showed that the mRNA expression levels of inflammation-related genes were significantly negative related to SelW (p?相似文献   

Down-Regulation of Adiponectin Receptors in Osteoarthritic Chondrocytes

The objective of this study was to investigate the expression of adiponectin receptors (AdipoR1, R2, and T-cadherin) in both normal subjects and patients with knee osteoarthritis (OA). We used immunofluorescence to assess expression of adiponectin receptors in the chondrocytes of normal subjects (n = 3) and OA patients (n = 3). We also studied mRNA expression of adiponectin receptors in both groups by real-time polymerase chain reaction (real-time PCR). Finally, we utilized Western blotting to confirm the presence of adiponectin receptors. As compared with osteoarthritic chondrocytes, normal chondrocytes showed stronger immunoreactivity for AdipoR1, AdipoR2, and T-cadherin. The expression levels of both AdipoR1 and AdipoR2 mRNA were significantly lower in the osteoarthritic chondrocytes compared with those in the normal chondrocytes, 19 ± 2 and 36 ± 3 % of normal chondrocytes, respectively (P < 0.001). T-cadherin mRNA expression levels of the osteoarthritic chondrocytes were also lower than those in the normal chondrocytes, but not statistical significant (P = 0.072). The expression levels of AdipoR1 and AdipoR2 protein were significantly higher in the normal chondrocytes compared with those in the osteoarthritic chondrocytes (P < 0.001, P < 0.01, respectively). T-cadherin protein expression level of the normal chondrocytes was also higher than those in the osteoarthritic chondrocytes, but the difference is not statistical significant (P = 0.114). Expression of adiponectin receptors protein in normal and osteoarthritic chondrocytes is consistent with its mRNA expression levels. In conclusion, we report for the first time down-regulation of adiponectin receptors (AdipoR1, R2, and T-cadherin) in osteoarthritic chondrocytes. Decreased adiponectin receptors in OA may reduce the tissue sensitivity to adiponectin, thus lost the protection from adiponectin in the progression of OA.     

A Systems Biology Approach to Study the Biology Characteristics of Esophageal Squamous Cell Carcinoma by Integrating microRNA and Messenger RNA Expression Profiling

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors. The aim of this study was to investigate the biology characteristics of ESCC by analyzing microRNA and mRNA expression profile. We used BRB-array tools to analyze the deregulated microRNA and mRNA between esophageal squamous cell carcinomas and paired normal adjacent tissues. We used miRTrail and protein–protein interaction methods to explore the related pathways and networks of deregulated microRNA and mRNA. By combining the results of pathways and networks, we found that the deregulated microRNA and their deregulated target mRNA are enriched in the following pathways: DNA replication, cell cycle, ECM-receptor interaction, focal adhesion, mismatch repair, and pathways in cancer. The results showed that many deregulated microRNAs and mRNAs may play a vital role in the pathogenesis of ESCC, and the systems biology approach is very helpful to explore molecular mechanism of ESCC.     

Evidence of endoplasmic reticulum stress and liver inflammation in the American mink Neovison vison with benign hepatic steatosis

We investigated the presence of inflammatory signs in the progression of fatty liver disease induced by fasting. Sixty standard black American mink (Neovison vison) were fasted for 0, 1, 3, 5, or 7 days and one group for 7 days followed by re-feeding for 28 days. Liver sections were evaluated histologically and liver mRNA levels indicating endoplasmic reticulum (ER) stress, adipogenic transformation, and inflammation were assessed by quantitative real-time PCR. After 3 days of fasting, the mink had developed moderate liver steatosis. Increased hyaluronan reactivity in lymphocytic foci but no Mallory–Denk bodies were seen in livers of the mink fasted for 5–7 days. Up-regulation of glucose-regulated protein, 78 kDa was observed on day 7 indicating ER stress, especially in the females. Liver lipoprotein lipase and monocyte chemoattractant protein 1 mRNA levels increased in response to 5–7 days of food deprivation, while tumor necrosis factor α (TNF-α) was the highest in the mink fasted for 5 days. The expression of the genes of interest, except for TNF-α, correlated with each other and with the liver fat content. The mRNA levels were found to change more rapidly below n-3/n-6 polyunsaturated fatty acid ratio threshold of 0.15. Following re-feeding, hepatocyte morphology and mRNA abundance returned to pre-fasting levels. Within the studied timeframe, evidence for ER stress, adipogenic transformation, and liver inflammation suggested incipient transition from steatosis to steatohepatitis with potential for development of more severe liver disease. This may present a possibility to influence disease progression before histologically observable steatohepatitis.     

PGC-1α is associated with C2C12 Myoblast differentiation

PGC-1α has been implicated as an important mediator of functional capacity of skeletal muscle. However, the role of PGC-1α in myoblast differentiation remains unexplored. In the present study, we observed a significant up-regulation of PGC-1α expression during the differentiation of murine C2C12 myoblast. To understand the biological significance of PGC-1α up-regulation in myoblast differentiation, C2C12 cells were transfected with murine PGC-1α cDNA and siRNA targeting PGC-1α, respectively. PGC-1α over-expressing clones fused to form typical myotubes with higher mRNA level of myosin heavy chain isoform I (MyHCI) and lower MyHCIIX. No obvious differentiation was observed in PGC-1α-targeted siRNA-transfected cells with marked decrement of mRNA levels of MyHCI and MyHCIIX. Furthermore, PGC-1α increased the expression of MyoD and MyoG in C2C12 cells, which controlled the commitment of precursor cells to myotubes. These results indicate that PGC-1α is associated with myoblast differentiation and elevates MyoD and MyoG expression levels in C2C12 cells.     

Expression of genes in the 111.35–116.16 million bp fragment of chromosome 13 in brain of mice with different predisposition to hereditary catalepsy

Catalepsy is a pathological animal behavior that is usually associated with dysfunctions in the striatal pallidal system of the brain and can be caused by different reasons. It was previously demonstrated that hereditary catalepsy is linked to the 111.35–116.16 million bp fragment of chromosome 13 in mice. The level of mRNA content in 42 genes localized in this fragment was determined in the study. Two brain departments that are functionally associated with catalepsy (striatum and substantia nigra) were studied in mice from AKR line (resistant to catalepsy), cataleptic CBA line, and recombinant cataleptic AKR.CBA-D13Mit76 (D13) line. The latter was obtained by the transfer of indicated fragment of chromosome 13 from the CBA line to the genome of the AKR line. It was found that two genes (Ndufs4 and Ppap2a) in the striatum and ten genes (Esm1, Fst, Gm10735, Gm15322, Gm15323, Gm15324, Gm15325, Il6st, Il31ra, and Itga1) in the substantia nigra differ in the level of mRNA expression in AKR and D13 lines. The Mcidas gene mRNA level is lower in both structures in D13 line mice than in the AKR line. The expression of the Hspb3 and Mocs2 genes (that encode heat shock protein and molybdenum cofactor synthesis, respectively) is lower in the substantia nigra of CBA and D13 cataleptic line mice than in the AKR line resistant to catalepsy. These genes are considered to be the most likely candidate genes of the catalepsy. The coexpression of a large amount of genes in these brain structures in sick animals indicates the existence of a complex gene network that regulates hereditary catalepsy.     

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.     

Loss of EphB6 protein expression in human colorectal cancer correlates with poor prognosis

Erythropoietin-producing hepatocyte (Eph) receptor family constitutes the largest family of tyrosine kinase receptors in the human genome. Loss of EphB6, a kinase-deficient receptor, correlated with a negative outcome in several carcinomas. This study aimed to investigate the expression of EphB6 protein and mRNA levels in colorectal cancers (CRCs) and possible correlations with clinicopathological variables and prognosis. To assess protein expression level, 124 CRCs and 57 colorectal adenomas samples were examined by immunostaining, the mRNA level of 43 paired CRC and the adjacent normal tissues were detected by using SYBR Green real-time PCR method. Decreased expression of EphB6 protein was found in CRC as compared with adenoma and normal tissues (χ² = 10.146, P = 0.001 and χ² = 45.333, P < 0.001, respectively). Low EphB6 mRNA expression was detected in 83.8 % of cancers with negative or low EphB6 protein expression. The loss of EphB6 protein in CRC was positively associated with poorly differentiation (P < 0.001), lymph node metastasis (P = 0.006), Dukes stage (P = 0.002) and depth of invasion (P = 0.016). The patients with lymph node metastasis had a worse prognosis independently of gender, age, tumor site, stage and differentiation (RR = 0.404, CI 0.267–0.213, P < 0.001). Low levels of EphB6 protein expression are associated with a shorter mean duration of survival in colorectal cancer. Our results demonstrated that EphB6 may represent a novel, useful tissue biomarker for the prediction of survival rate in CRC.     

Adenosylmethionine Decarboxylase 1 (AMD1)-Mediated mRNA Processing and Cell Adhesion Activated & Inhibited Transition Mechanisms by Different Comparisons Between Chimpanzee and Human Left Hemisphere

To understand adenosylmethionine decarboxylase 1 (AMD1)-mediated mRNA processing and cell adhesion activated & inhibited transition mechanisms between chimpanzee and human left hemisphere, AMD1-activated different complete (all no positive correlation, Pearson correlation coefficient < 0.25) and uncomplete (partly no positive correlation except AMD1, Pearson < 0.25) networks were identified in higher human compared with lower chimpanzee left hemisphere from the corresponding AMD1-stimulated (Pearson ≥ 0.25) or inhibited (Pearson ≤ ?0.25) overlapping molecules of Pearson and GRNInfer, respectively. This result was verified by the corresponding scatter matrix. As visualized by GO, KEGG, GenMAPP, BioCarta, and disease database integration, we proposed mainly that AMD1-stimulated different complete network was involved in AMD1 activation with cytoplasm ubiquitin specific peptidase (tRNA-guanine transglycosylase) to nucleus paired box-induced mRNA processing, whereas the corresponding inhibited network participated in AMD1 repression with cytoplasm protocadherin gamma and adaptor-related protein complex 3-induced cell adhesion in lower chimpanzee left hemisphere. However, AMD1-stimulated network contained AMD1 activation with plakophilin and phosphodiesterase to SH3 binding glutamic acid-rich protein to dynein and zinc finger-induced cell adhesion, whereas the corresponding inhibited different complete network included AMD1 repression with mitochondrial denine nucleotide translocator, brain protein, and ADH dehydrogenase to ribonucleoprotein-induced mRNA processing in higher human left hemisphere. Our AMD1 different networks were verified by AMD1-activated or -inhibited complete and uncomplete networks within and between chimpanzee left hemisphere or (and) human left hemisphere.     

PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer

Polo-like kinase 4 (PLK4) is a centrosomal protein that is involved in the regulation of centrosome duplication. This study aimed to determine whether the genetic abnormality of PLK4 is involved in human gastric cancer. First, we examined the status of PLK4 mRNA expression in 7 gastric cancer cell lines and 48 primary gastric cancers using an RT-PCR analysis. The upregulation of PLK4 mRNA expression was detected in 57.1 % (4/7) of the gastric cancer cell lines, and a novel PLK4 variant with exon 4, but without exon 5, was identified. In the primary gastric cancers, the upregulation of PLK4 mRNA expression in the cancerous cells was detected in 50.0 % (24/48) of the cases, and this upregulation was statistically significant (P value = 0.0139). Next, we established AGS gastric cancer cells capable of inducibly expressing PLK4 using the piggyBac transposon vector system and showed that PLK4 overexpression induced centrosome amplification and chromosome instability using immunofluorescence and FISH analyses, respectively. Furthermore, PLK4 overexpression suppressed primary cilia formation. Our current findings suggested that PLK4 is upregulated in a subset of primary gastric cancers and that PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.     

Expression and functional analysis of putative vacuolar Ca2+-transporters (CAXs and ACAs) in roots of salt tolerant and sensitive rice cultivars

Vacuolar Ca²⁺-transporters could play an important role for salt tolerance in rice (Oryza sativa L.) root. Here, we compared the expression profiles of putative vacuolar cation/H⁺ exchanger (CAX) and calmodulin-regulated autoinhibited Ca²⁺-ATPase (ACA) in rice roots of salt tolerant cv. Pokkali and salt sensitive cv. IR29. In addition to five putative vacuolar CAX genes in the rice genome, a new CAX gene (OsCAX4) has been annotated. In the present study, we isolated the OsCAX4 gene and showed that its encoded protein possesses a unique transmembrane structure and is potentially involved in transporting not only Ca²⁺ but also Mn²⁺ and Cu²⁺. These six OsCAX genes differed in their mRNA expression pattern in roots of tolerant versus sensitive rice cultivars exposed to salt stress. For example, OsCAX4 showed abundant expression in IR29 (sensitive) upon prolonged salt stress. The mRNA expression profile of four putative vacuolar Ca²⁺-ATPases (OsACA4-7) was also examined. Under control conditions, the mRNA levels of OsACA4, OsACA5, and OsACA7 were relatively high and similar among IR29 and Pokkali. Upon salt stress, only OsACA4 showed first a decrease in its expression in Pokkali (tolerant), followed by a significant increase. Based on these results, a role of vacuolar Ca²⁺ transporter for salt tolerance in rice root was discussed.