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Quantitative real-time reverse transcription-polymerase chain reaction (Q-PCR) has become an indispensable technique for accurate determination of gene expression in various samples. In mice, intravenous injection of concanavalin A (ConA) leads to acute hepatitis and liver injury. Functional studies based on this model have provided insights for understanding the mechanisms of liver injury. However, no data have been reported to validate reference genes during the progression of ConA-induced hepatitis (CIH). In this study, IkappaBalpha and C/EBPbeta messenger RNA (mRNA) levels were examined using Q-PCR with ACTB as the reference gene after ConA injection. However, we got inconsistent results with previous reports determining IkappaBalpha and C/EBPbeta mRNA expression levels. The results indicate the necessity for stability analysis of candidate reference genes in the CIH model. geNorm, NormFinder, and BestKeeper software analysis indicates that ACTB is the most unstable gene during CIH progression among the 10 reference genes tested, whereas RPLP0 or HPRT1 is the most stable one. This study demonstrates that some of the commonly used reference genes are inadequate for normalization of Q-PCR data due to their expression instability. Furthermore, this study validates HPRT1 and RPLP0 as appropriate reference genes for Q-PCR analysis in the CIH model., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: Obesity contributes to many common diseases, including cardiovascular and metabolic disorders such as diabetes, hypertension, and stroke. Leptin and ciliary neurotrophic factor (CNTF), two members of the cytokine family, play important roles in controlling food intake and body weight in rodents. Here, we used the hydrodynamics-based gene delivery technique to introduce leptin and CNTF expression plasmids, pCAGGS-leptin and pCAGGS-CNTF, into mice and to assess whether they could induce high expression of leptin or CNTF and reduce food intake and body weight in the mice., Methods: Plasmid DNA (50 microg) in lactated Ringer's solution (0.1 ml/g body weight) was rapidly injected into the tail vein of 4-week-old male MCH-ICR mice., Results: After a single injection, serum leptin peaked (at 32.0 +/- 3.1 ng/ml) 2 days after the injection, and serum CNTF peaked (at 22.0 +/- 0.8 microg/ml) 1 day after the injection. The high expression of either leptin or CNTF was sustained and dramatically reduced food intake (CNTF and leptin group vs. control group; 2.3 +/- 0.5 and 3.1 +/- 0.5 g/day vs. 4.8 +/- 0.6 g/day; p < 0.001) and body weight (3 weeks after the injection; CNTF and leptin group vs. control group; -19.5% and +3.3% vs. +33.3-47.5%; p < 0.001) in the mice, suggesting potent in vivo activities for these exogenously expressed proteins., Conclusions: These results suggest that hydrodynamics-based gene delivery could have broad applications in the study of appetite regulation and metabolism., (Copyright 2003 John Wiley & Sons, Ltd.)

Summary: R‐loops, three‐stranded nucleic acid structures consisting of a DNA: RNA hybrid and displaced single‐stranded DNA, play critical roles in gene expression and genome stability. How R‐loop homeostasis is integrated into chloroplast gene expression remains largely unknown.We found an unexpected function of FtsHi1, an inner envelope membrane‐bound AAA‐ATPase in chloroplast R‐loop homeostasis of Arabidopsis thaliana.Previously, this protein was shown to function as a component of the import motor complex for nuclear‐encoded chloroplast proteins. However, this study provides evidence that FtsHi1 is an ATP‐dependent helicase that efficiently unwinds both DNA–DNA and DNA–RNA duplexes, thereby preventing R‐loop accumulation. Over‐accumulation of R‐loops could impair chloroplast transcription but not necessarily genome integrity. The dual function of FtsHi1 in both protein import and chloroplast gene expression may be important to coordinate the biogenesis of nuclear‐ and chloroplast‐encoded subunits of multi‐protein photosynthetic complexes.This study suggests a mechanical link between protein import and R‐loop homeostasis in chloroplasts of higher plants. [ABSTRACT FROM AUTHOR]

Abstract Hemorrhagic septicemia of mandarin fish (Siniperca chuatsi) was mainly caused by Aeromonas hydrophila which was an opportunistic pathogen. In recent years, the disease has caused tremendous economic loss with high morbidity and mass mortality in the mandarin fish breeding industry. Histopathological analysis and the immune related gene expression profiles of mandarin fish (S. chuatsi) infected with A. hydrophila were investigated in this study. Transmission electron microscopy (TEM) images showed that the cells of A. hydrophila densely covered with a mass of fimbriae. Histopathological analysis revealed that inflammation, vacuolization and extensive necrosis existed in the gill, liver, spleen and head kidney of the diseased fish. Quantitative real-time PCR was performed to measure mRNA expression levels for six immune related genes in mandarin fish after A. hydrophila infection. The transcriptional analysis of these immune related genes demonstrated that the expression levels of major histocompatibility complex class II (MHC II), T cell receptor α (TCRα), tumor necrosis factor α (TNFα), CC chemokine 3, interleukin 8 (IL-8) and Hepcidin were strongly up-regulated in spleen and head kidney of mandarin fish post-infection. These results will contribute to further study on the pathogenesis and host defensive system in A. hydrophila infection. Highlights • The morphological characteristic of Aeromonas hydrophila isolated from the diseased mandarin fish was observed. • Histopathological analysis related to mandarin fish with Aeromonas hydrophila infection were studied. • Aeromonas hydrophila triggers a wide defensive response of various immune related genes in mandarin fish. [ABSTRACT FROM AUTHOR]

Background: About 2% of pregnant women receive non-obstetric surgery under general anesthesia each year. During pregnancy, general anesthetics may affect brain development of the fetus. This study aimed to investigate safe dosage range of isoflurane. Methods: Forty-eight SpragueDawley (SD) pregnant rats were randomly divided into 3 groups and inhaled 1.3% isoflurane (the Iso1 group), 2.0% isoflurane (the Iso2 group) and 50% O2 alone (the control group) for 3 h, respectively. Their offsprings were subjected to Morris water maze at day 28 and day 90 after birth to evaluate learning and memory. The expression of cAMP-response element binding protein (CREB) and phosphorylated cAMP-response element binding protein (p-CREB) was detected in the hippocampus dentate gyrus. Results: Less offsprings of Iso2 group were able to cross the platform than that of the control group (P < 0.05). Accordingly, the Iso2 offsprings expressed p-CREB mainly in the subgranular zone in contrast to the whole granular cell layer of hippocampus dentate gyrus as detected in the Iso1 and control offsprings; the expression level of pCREB was also lower in the Iso2 than Iso1 or control offsprings (P < 0.05). Conclusion: Inhalation of isoflurane at 1.3% during pregnancy has no significant influence on learning and memory of the offspring; exposure to isoflurane at 2.0% causes damage to spatial memory associated with inhibition of CREB phosphorylation in the granular cell layer of hippocampus dentate gyrus. [ABSTRACT FROM AUTHOR]

Mouse models have been widely utilized to elucidate the basic principles and regulatory mechanisms of primordial follicle activation. Outside their natural environment, the growth of follicles might be affected by unknown factors in vitro and the elimination of regulation in vivo. Currently, in vitro culture and transplantation of ovaries under the kidney capsule are two commonly used incubation methods. However, the limited number of studies that have been published compare various incubation systems and reveal differences between ovaries that are incubated and grown in vivo. We compare the number of primordial, primary and secondary follicles in cultured, transplanted and in-vivo-grown ovaries. We investigate the expression levels of four genes, including zona pellucida 3 (ZP3), growth and differentiation factor-9 (GDF-9), proliferating cell nuclear antigen (PCNA) and anti-Müllerian hormone (AMH). Our results suggest that in vitro culture accelerates follicle activation, delays the transition from primary to secondary follicles and affects the expression patterns of ZP3, GDF-9, PCNA and AMH. A larger number of secondary follicles in ovaries cultured in alpha-minimal essential medium (α-MEM) had intact zona pellucida compared with those grown in Dulbecco's modified Eagle medium containing Ham's F-12 nutrient mixture (D/F12), suggesting that α-MEM is a better basal medium. The transplanted ovaries demonstrated the most similar characteristics to the in-vivo-grown ovaries, indicating that transplantation provided an optimal environment for ovarian incubation. This study has thus established the similarities and differences between in-vivo-grown and incubated ovaries, demonstrated that transplantation can mostly mimic the environment of ovarian growth in vivo and determined the optimal basal culture medium between α-MEM and D/F12. [ABSTRACT FROM AUTHOR]

Fertilization is controlled by a complex gene regulatory network. To study the fertilization mechanism, we determined time courses of the four developmental stages of fertilization in Chinese cabbage pak-choi ( Brassica campestris subsp. chinensis) by cytological observation. We then used the Arabidopsis ATH1 microarray to characterize the gene expression profiles of pollinated and unpollinated pistils in B. campestris subsp. chinensis. The result showed 44 up-regulated genes and 33 down-regulated genes in pollinated pistils compared with unpollinated pistils. Gene ontology analysis identified 20% of the up-regulated genes as belonging to the category of cell wall metabolism. We compared the up-regulated genes in pollinated pistils with previously identified pollen development related genes. Ten genes were found to be in common, which were termed as continuously expressed genes, in the two processes in the present article. Their expression patterns during pollen development and fertilization processes were then verified by RT-PCR. One of the continuously expressed genes, the homologous gene of At3g01270 in B. campestris subsp. chinensis, was confirmed as specifically expressed in microspores and pollinated pistils by using in situ hybridization. The potential biological functions of the other continuously expressed genes were also discussed. [ABSTRACT FROM AUTHOR]

Abstract: B-type natriuretic peptide (BNP) and its related peptides are biomarkers for the diagnosis of heart failure. Recent studies identified several O-glycosylation sites, including Thr-71, on human pro-BNP but the functional significance was unclear. In this study, we analyzed glycosylation and proteolytic processing of pro-BNP in cardiomyocytes. Human pro-BNP wild-type (WT) and mutants were expressed in HEK 293 cells and murine HL-1 cardiomyocytes. Pro-BNP and BNP were analyzed by immunoprecipitation and Western blotting. Glycosidases and glycosylation inhibitors were used to examine carbohydrates on pro-BNP. The effects of furin and corin expression on pro-BNP processing in cells also were examined. We found that in HEK 293 cells, recombinant pro-BNP contained significant amounts of O-glycans with terminal oligosialic acids. Mutation at Thr-71 reduced O-glycans on pro-BNP and increased pro-BNP processing. In HL-1 cardiomyocytes, residue Thr-71 contained little O-glycans, and pro-BNP WT and T71A mutant were processed similarly. In HEK 293 cells, pro-BNP was processed by furin. Mutations at Arg-73 and Arg-76, but not Lys-79, prevented pro-BNP processing. In HL-1 cardiomyocytes, which express furin and corin, single or double mutations at Arg-73, Arg-76 and Lys-79 did not prevent pro-BNP processing. Only when all these three residues were mutated, was pro-BNP processing completely blocked. Our data indicate that pro-BNP glycosylation in cardiomyocytes differed significantly from that in HEK 293 cells. In HEK 293 cells, furin cleaved pro-BNP at Arg-76 whereas in cardiomyocytes corin cleaved pro-BNP at multiple residues including Arg-73, Arg-76 and Lys-79. [Copyright &y& Elsevier]

• This study provided a comprehensive analysis of the underlying mechanisms of EMT in CD. • Compared with Control group, there was more infiltration of M1 macrophages and less infiltration of M2 macrophages in CD group. • Two machine learning approaches were used to screen the key genes LCN2 and LPCAT1. • The effects of LPCAT1 on EMT and fibrosis were verified in LPS-induced HT-29 cells. Epithelial-mesenchymal transition (EMT) plays a crucial role in regulating inflammatory responses and fibrosis formation. This study aims to explore the molecular mechanisms of EMT-related genes in Crohn's disease (CD) through bioinformatics methods and identify potential key biomarkers. In our research, we identified differentially expressed genes (DEGs) related to EMT based on the GSE52746 dataset and the gene set in the GeneCards database. Key genes were identified through Lasso-cox and Random Forest and validated using the external dataset GSE10616. Immune infiltration analysis showed that Lysophosphatidylcholine acyltransferase 1 (LPCAT1) was positively correlated with Neutrophils and Macrophages M1. The Gene Set Enrichment Analysis (GSEA) results for LPCAT1 showed associations with cell adhesion molecules and ECM receptor interaction. Additionally, a lncRNA-miRNA-mRNA ceRNA network was constructed. Finally, we validated that knocking down LPCAT1 could inhibit the release of inflammatory factors, EMT, and the elevation of fibrosis indices as well as the activation of NF-κB signaling pathway in LPS-induced HT-29 cells. LPCAT1 plays an important role in the occurrence and development of CD and may become a new biomarker. [ABSTRACT FROM AUTHOR]

Abstract: The clip-domain serine proteases (SPs) are the essential components of extracellular signaling cascade in various biological processes, especially in embryonic development and the innate immune responses of invertebrate. Herein, we described the isolation and characterization of pearl oyster Pinctada fucata clip-domain SP gene (designated as poSP). The poSP cDNA was 1080bp long and consisted of a 5′-untranslated region (UTR) of 13bp, a 3′-UTR of 68bp with a polyadenylation signal (AATAAA) at 22 nucleotides upstream of the poly(A) tail, and an open reading frame (ORF) of 999bp encoding a polypeptide of 332 amino acids with an estimated molecular mass of 36.5kDa and a theoretical isoelectric point of 7.3. A clip-domain and a trypsin-like serine protease domain were identified in the poSP using SMART analysis. Homology analysis of the deduced amino acid sequence of the poSP with other known SP sequences by MatGAT software revealed that the poSP shared 47.0–68.4% similarity to the other known SP sequences. The poSP mRNA was expressed in haemocytes, gonad, digestive gland and mantle, but not expressed in adductor muscle and gill. The poSP mRNA was up-regulated and increased nearly double-fold after LPS or Vibrio alginolyticus stimulation, respectively. These results suggested that the poSP was an inducible acute-phase protein that perhaps involved in the innate immune response of pearl oyster. [Copyright &y& Elsevier]