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Mimecan encodes a secretory protein that is secreted into the human serum as two mature proteins with molecular masses of 25 and 12 kDa. We found 12-kDa mimecan to be a novel satiety hormone mediated by the upregulation of the expression of interleukin (IL)-1β and IL-6 in the hypothalamus. Mimecan was found to be expressed in human pituitary corticotroph cells and was up-regulated by glucocorticoids, while the secretion of adrenocorticotropic hormone (ACTH) in pituitary corticotroph AtT-20 cells was induced by mimecan. However, the effects of mimecan in adrenal tissue on the hypothalamic-pituitary-adrenal (HPA) axis functions remain unknown. We demonstrated that the expression of mimecan in adrenal tissues is significantly downregulated by hypoglycemia and scalded stress. It was down-regulated by ACTH, but upregulated by glucocorticoids through in vivo and in vitro studies. We further found that 12-kDa mimecan fused protein increased the corticosterone secretion of adrenal cells in vivo and in vitro. Interestingly, compared to litter-mate mice, the diurnal rhythm of corticosterone secretion was disrupted under basal conditions, and the response to restraint stress was stronger in mimecan knockout mice. These findings suggest that mimecan stimulates corticosterone secretion in the adrenal tissues under basal conditions; however, the down-regulated expression of mimecan by increased ACTH secretion after stress in adrenal tissues might play a role in maintaining the homeostasis of an organism's responses to stress.

Paraquat (PQ) as a non-selective heterocyclic herbicide, has been applied worldwide for over a few decades. But PQ is very harmful to humans and rodents. The lung is the main target organ of PQ poisoning. It is an important event that lung epithelial cells are injured during PQ-induced acute lung injury and pulmonary fibrosis. As a regulator of mRNA expression, microRNA (miRNA) may play an important role in the progress. Our study was to investigate the mechanisms of PQ-induced injury of pulmonary epithelial cells through analyzing the profiling of miRNAs and their target genes. As a result, 11 differentially expressed miRNAs were screened, including 1 upregulated miRNA and 10 downregulated miRNAs in PQ-treated murine lung alveolar epithelial cells (MLE-12 cells). The bioinformatic analyses suggested that the target genes of these miRNAs were involved in mitochondrial apoptosis pathway and DNA methylation, and participated in the regulation of PI3K-Akt, mTOR, RAS, TNF, MAPK and other signal pathways which related to oxidative stress and apoptosis. This indicated that miRNAs were an important regulator of oxidative stress and apoptosis during PQ-induced injury of murine lung alveolar epithelial cells. The findings would deepen our understanding of the mechanisms of PQ-induced pulmonary injury and might provide new treatment targets for this disease.

e14654 Background: Formalin-fixed and paraffin-embedded (FFPE) blocks held in clinical laboratories are an invaluable resource for clinical research, especially in the era of personalized medicine. With the development of massive parallel sequencing, RNA expression profiles have become an important source of new biomarkers with potential values in cancer metastasis and disease prognosis. Standard practice in tissue fixing and paraffin-embedding has little impact on the expression analysis of RNA samples, which makes archived FFPE samples valuable and retrospective studies feasible. But it is still difficult for some FFPE samples which have low quality or low amount of RNA to be used in the RNA expression study. Methods: We compared four FFPE RNA library preparation kits (KAPA, TaKaRa, QIAGEN and Vazyme) based on two principles of rRNA depletion, with total RNA from FFPE samples and paired FF samples using two bioinformatics analysis methods. Furthermore, we tried to construct RNA libraries with the two kits allowing for degraded low-input RNA using clinical FFPE samples of different quality. HISAT was used to analyze the data of all the libraries. Results: Our results demonstrated that high concordance in transcript quantifications were shown between FF and FFPE samples using the same kit and more transcripts could be obtained by rRNA depletion after reverse transcript reaction. The gene expression profiling using the TaKaRa kit showed a difference with other kits, which may be due to the different principle of rRNA depletion or the amount of input total RNA. For seriously degraded RNA from FFPE samples, libraries could be constructed with as low as 50 ng of total RNA, although there was higher residual rRNA and lower unique mapping ratio in the libraries. Conclusions: These results indicated that most of clinical FFPE samples could be used in the RNA profiling study. The method of RNA library construction with rRNA depletion can be used for clinical FFPE samples. For degraded and low-input RNA samples, it is still possible to obtain repeatable RNA expression profiling but with a low unique mapping ratio and high residual rRNA.

Extrachromosomal DNA (ecDNA) is a type of circular and tumor specific genetic element. EcDNA has been reported to display open chromatin structure, facilitate oncogene amplification and genetic material unequal segregation, and is associated with poor cancer patients’ prognosis. The ability of immune evasion is a typical feature for cancer progression, however the tumor intrinsic factors that determine immune evasion remain poorly understood. Here we show that the presence of ecDNA is associated with markers of tumor immune evasion, and obtaining ecDNA could be one of the mechanisms employed by tumor cells to escape immune surveillance. Tumors with ecDNA usually have comparable TMB and neoantigen load, however they have lower immune cell infiltration and lower cytotoxic T cell activity. The microenvironment of tumors with ecDNA shows increased immune-depleted, decreased immune-enriched fibrotic types. Both MHC class I and class II antigen presentation genes’ expression are decreased in tumors with ecDNA, and this could be the underlying mechanism for ecDNA associated immune evasion. This study provides evidence that ecDNA formation is an immune escape mechanism for cancer cells.

Early-life antibiotic exposure perturbs the intestinal microbiota and accelerates type 1 diabetes (T1D) development in the NOD mouse model. Here, we found that maternal cecal microbiota transfer (CMT) to NOD mice after early-life antibiotic perturbation largely rescued the induced T1D enhancement. Restoration of the intestinal microbiome was significant and persistent, remediating the antibiotic-depleted diversity, relative abundance of particular taxa, and metabolic pathways. CMT also protected against perturbed metabolites and normalized innate and adaptive immune effectors. CMT restored major patterns of ileal microRNA and histone regulation of gene expression. Further experiments suggest a gut-microbiota-regulated T1D protection mechanism centered on Reg3γ, in an innate intestinal immune network involving CD44, TLR2, and Reg3γ. This regulation affects downstream immunological tone, which may lead to protection against tissue-specific T1D injury.

To explore the possible roles of metallothioneins (MTs) played in cadmium (Cd) accumulation of water spinach, three IaMT genes, IaMT1, IaMT2 and IaMT3 in a high-shoot-Cd (T308) and a low-shoot-Cd accumulation cultivar (QLQ) were cloned, characterized, and quantitated. Gene expression analysis suggested that the expression of the IaMTs was differentially regulated by Cd stress in different cultivars, and T308 showed higher MTs expression overall. Furthermore, only shoot IaMT3 expression was cultivar dependent among the three IaMTs. Antioxidant analysis showed that the high production of IaMTs in T308 should be associated with its high oxidation resistance. The role of IaMTs in protecting against Cd toxicity was demonstrated in vitro via recombinant E. coli strains. The results showed that IaMT1 correlated with neither Cd tolerance nor Cd accumulation of E. coli, while IaMT2 conferred Cd tolerance in E. coli, IaMT2 and IaMT3 increased Cd accumulation in E. coli. These findings help to clarify the roles of IaMTs in Cd accumulation, and increase our understanding of the cultivar-dependent Cd accumulation in water spinach.

Primary human hepatocytes (PHHs) are the 'gold standard' for investigating hepatitis B virus (HBV) infection and antiviral drugs. However, poor availability, variation between batches and ethical issues regarding PHHs limit their applications. The discovery of human sodium taurocholate co‑transporting polypeptide (hNTCP) as a functional HBV receptor has enabled the development of a surrogate model to supplement the use of PHHs. In the present study, the evolutionary distance of seven species was assessed based on single‑copy homologous genes. Based on the evolutionary distance and availability, PHHs and primary rabbit hepatocytes (PRHs) were isolated and infected with hNTCP‑recombinant lentivirus, and susceptibility to HBV infection in the two cell types was tested and compared. In addition, HBV infection efficiency of hNTCP‑expressing PPHs with pooled HBV‑positive serum and purified particles was determined. The potential use of HBV‑infected hNTCP‑expressing PPHs for drug screening was assessed. The results demonstrated that pigs and rabbits are closer to humans in the divergence tree compared with mice and rats, indicating that pigs and rabbits were more likely to facilitate the HBV post‑entry lifecycle. Following hNTCP complementation and HBV infection, PPHs and Huh7D human hepatocellular carcinoma cells, but not PRHs, exhibited increased hepatitis B surface antigen and hepatitis B e‑antigen secretion, covalently closed circular DNA formation and infectious particle secretion. hNTCP‑expressing PPHs were susceptible to infection with HBV particles purified from pooled HBV‑positive sera, but were poisoned by raw HBV‑positive sera. The use of HBV‑infected hNTCP‑expressing PPHs for viral entry inhibitor screening was revealed to be applicable and reproducible. In conclusion, hNTCP‑expressing PPHs may be valuable tool for investigating HBV infection and antiviral drugs.

The exposure to combinations of heavy metals can affect the genes of vegetables and heavy metals would accumulate in vegetables and thereby indirectly affecting human health. Exploring the links between genetic changes and phenotypic changes of carrot under the combined pollution of Cd and Cu is of great significance for studying the mechanism of heavy metal pollution. Therefore, this study examined the effects of mixtures of cadmium (Cd) and copper (Cu) on physiological measures (malondialdehyde (MDA), proline, and antioxidant enzyme) and expression of growth-related genes (gibberellin gene, carotene gene, and glycogene) in carrot under greenhouse cultivation. The results showed in the additions with mixtures of Cd and Cu at higher concentration, the MDA content increased significantly (p 0.05), whereas the proline content was not significantly different from those in the control. In the mixed treatments with high Cd concentrations, the activity of superoxide dismutase (SOD) was significantly lower than that in the control (p 0.05); whereas the activity of peroxidase (POD) increased to different degrees compared to the control. In the additions with mixtures of Cd and Cu, compared with the control, the expression of the gibberellin gene was downregulated from 1.97 to 20.35 times (not including the 0.2 mg kg

Adipokines such as leptin play important roles in the regulation of energy metabolism, particularly in the control of appetite. Here, we describe a hormone, mimecan, which is abundantly expressed in adipose tissue. Mimecan was observed to inhibit food intake and reduce body weight in mice. Intraperitoneal injection of a mimecan-maltose binding protein (-MBP) complex inhibited food intake in C57BL/6J mice, which was attenuated by pretreatment with polyclonal antibody against mimecan. Notably, mimecan-MBP also induced anorexia in Ay/a and db/db mice. Furthermore, the expression of interleukin (IL)-1β and IL-6 was up-regulated in the hypothalamus by mimecan-MBP, as well as in N9 microglia cells by recombinant mouse mimecan. Taken together, the results suggest that mimecan is a satiety hormone in adipose tissue, and that mimecan inhibits food intake independently of leptin signaling by inducing IL-1β and IL-6 expression in the hypothalamus.

The early-life intestinal microbiota plays a key role in shaping host immune system development. We found that a single early-life antibiotic course (1PAT) accelerated type 1 diabetes (T1D) development in male NOD mice. The single course had deep and persistent effects on the intestinal microbiome, leading to altered cecal, hepatic, and serum metabolites. The exposure elicited sex-specific effects on chromatin states in the ileum and liver and perturbed ileal gene expression, altering normal maturational patterns. The global signature changes included specific genes controlling both innate and adaptive immunity. Microbiome analysis revealed four taxa each that potentially protect against or accelerate T1D onset, that were linked in a network model to specific differences in ileal gene expression. This simplified animal model reveals multiple potential pathways to understand pathogenesis by which early-life gut microbiome perturbations alter a global suite of intestinal responses, contributing to the accelerated and enhanced T1D development., eLife digest The human body contains many microbes that play important roles in our health. These microbes begin to live in the intestines, skin, and mouth shortly after birth. They form complex communities called the microbiome, which changes as babies develop. The microbiome works with organs to maintain human health. For example, the lower intestinal tract is home to the most numerous and active microbes in the body. The intestines provide microbes with food and a welcoming environment, and the microbes make products the body needs, influence immune system development, and help maintain a balance of beneficial microbes. Use of antibiotics to treat infections, particularly early in life, disrupts intestinal microbe communities. Recent studies show that such microbiome disturbances may affect how the immune system develops and the rate at which type 1 diabetes develops. Type 1 diabetes is an autoimmune disease in which the immune system destroys cells in the pancreas that produce insulin. Scientists would like to learn more about how use of antibiotics in early life may contribute to the development of this disease. Now, Zhang et al. show that a single course of antibiotics administered early in life accelerates the development of type 1 diabetes in mice prone to develop the disease. In the experiments, a strain of laboratory mice that spontaneously develops type 1 diabetes were either given a single course of antibiotics, three courses of antibiotics, or no antibiotics in their first weeks of life. After one single course, the gut microbiome was different in mice treated with antibiotics compared with mice who were never exposed. The antibiotics also changed the molecules produced by these microbes. These alterations in the microbiome turned on or off certain genes in the intestine, affecting the development of the immune system. Zhang et al. identified some microbes that appear to protect against type 1 diabetes and others that seem to speed it up and how they do so. Antibiotic use in children is very common, so finding ways to reduce its potentially harmful effects on development are critical. The experiments provide one way to study how antibiotics may contribute to autoimmune disease. It also may allow scientists to test ways to reverse harmful change.

Background Gastric Helicobacter pylori colonization leads to iron deficiency anemia (IDA), especially in children and adolescents. However the pathogenesis is poorly understood. Objective We sought to identify specific H. pylori genes involved in IDA development, by comparing bacterial genome-wide expression profiling in patients affected or not. Methods H. pylori were isolated from four children with IDA and four from matched controls without IDA. Based on these isolates, cDNA microarrays under iron-replete or depleted conditions were systematically performed to compare gene expression profiles at the whole genome level. Real-time reverse-transcription (RT-) PCR and protein assays were performed for further assessing the profile differentiation of the identified H. pylori IDA-associated genes. Results We identified 29 and 11 genes with significantly higher or lower expression in the IDA isolates compared to non-IDA isolates, respectively. Especially notable were higher expression of sabA gene encoding sialic acid-binding adhesin in the IDA isolates, which was confirmed by real-time RT-PCR study. Moreover, iron-depletion in vitro led to up-regulation of fecA1 and frpB1 genes and down-regulation of pfr, as predicted. Known iron-regulated genes such as fur, pfr, fecA, and feoB did not significantly differ between both groups. The IDA isolates had significantly higher expression of vacuolating cytotoxin gene vacA than non-IDA isolates, consistent with the results of VacA protein assays. There were no significant differences in bacterial growth value between IDA and non-IDA isolates. Conclusions It is likely that H. pylori carrying high expression of sabA causes IDA, especially in children and adolescents who have increased daily iron demand. In addition, it is possible that several host-interactive genes, including vacA, may play a synergistic role for sabA in IDA development.

The rice blast, caused by fungus Magnaporthe oryzae , is a major constraint to the world food security. Hyphal growth is the foundation of fungal development and proliferation of fungi. To investigate genes involved in hyphal growth of this fungus, digital gene expression tag profiling was used to compare a previously generated temperature-sensitive mutant which defect at hyphae growth and reduction on pathogenicity, with its related wildtype strain. 416 genes were detected as differential expression, 178 of which were specifically expressed in Guy-11 but down-regulated expression in the mutant. Functional classification analysis revealed the phenotype mutation may be mainly caused by a defection in translational and vacuole-related processes. The results and the protocol used will improve our knowledge on morphogenesis and promote the further study on M. oryzae pathogenesis.

The early life microbiome plays important roles in host immunological and metabolic development. Because the incidence of type 1 diabetes (T1D) has been increasing substantially in recent decades, we hypothesized that early-life antibiotic use alters gut microbiota, which predisposes to disease. Using non-obese diabetic mice that are genetically susceptible to T1D, we examined the effects of exposure to either continuous low-dose antibiotics or pulsed therapeutic antibiotics (PAT) early in life, mimicking childhood exposures. We found that in mice receiving PAT, T1D incidence was significantly higher, and microbial community composition and structure differed compared with controls. In pre-diabetic male PAT mice, the intestinal lamina propria had lower Th17 and Treg proportions and intestinal SAA expression than in controls, suggesting key roles in transducing the altered microbiota signals. PAT affected microbial lipid metabolism and host cholesterol biosynthetic gene expression. These findings show that early-life antibiotic treatments alter the gut microbiota and its metabolic capacities, intestinal gene expression and T-cell populations, accelerating T1D onset in non-obese diabetic mice.

We have reported that the bacterial LPS induces the activation of NF-κB and inflammatory cytokine gene expression and that this requires the activity of small GTPase, RhoA. In this study, we show that an atypical protein kinase C isozyme, PKCζ, associates functionally with RhoA and that PKCζ acts as a signaling component downstream of RhoA. Stimulation of monocytes and macrophages with LPS resulted in PKCζ activation and that inhibition of PKCζ activity blocks both LPS-stimulated activation of NF-κB and IL-1β gene expression. Our results also indicate that transforming growth factor β-activated kinase 1 acts as a signaling component downstream of PKCζ in cytokine gene transcription stimulated by LPS in human peripheral blood monocytes and macrophages. The specificity of this response suggests an important role for the Rho GTPase/PKCζ/transforming growth factor β-activated kinase 1/NF-κB pathway in host defense and in proinflammatory cytokine synthesis induced by bacterial LPS.

We have shown that the quorum-sensing signals acylhomoserine lactones, autoinducer-2 (AI-2) and indole influence the biofilm formation of Escherichia coli. Here, we investigate how the environment, that is, temperature, affects indole and AI-2 signaling in E. coli. We show in biofilms that indole addition leads to more extensive differential gene expression at 30 degrees C (186 genes) than at 37 degrees C (59 genes), that indole reduces biofilm formation (without affecting growth) more significantly at 25 and 30 degrees C than at 37 degrees C and that the effect is associated with the quorum-sensing protein SdiA. The addition of indole at 30 degrees C compared to 37 degrees C most significantly repressed genes involved in uridine monophosphate (UMP) biosynthesis (carAB, pyrLBI, pyrC, pyrD, pyrF and upp) and uracil transport (uraA). These uracil-related genes are also repressed at 30 degrees C by SdiA, which confirms SdiA is involved in indole signaling. Also, compared to 37 degrees C, indole more significantly decreased flagella-related qseB, flhD and fliA promoter activity, enhanced antibiotic resistance and inhibited cell division at 30 degrees C. In contrast to indole and SdiA, the addition of (S)-4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor) leads to more extensive differential gene expression at 37 degrees C (63 genes) than at 30 degrees C (11 genes), and, rather than repressing UMP synthesis genes, AI-2 induces them at 37 degrees C (but not at 30 degrees C). Also, the addition of AI-2 induces the transcription of virulence genes in enterohemorrhagic E. coli O157:H7 at 37 degrees C but not at 30 degrees C. Hence, cell signals cause diverse responses at different temperatures, and indole- and AI-2-based signaling are intertwined.

Quorum-sensing signal autoinducer 2 (AI-2) stimulates Escherichia coli biofilm formation through the motility regulator MqsR that induces expression of the putative transcription factor encoded by yncC. Here, we show that YncC increases biofilm formation by repressing overproduction of the exopolysaccharide identified as colanic acid (corroborated by decreasing mucoidy and increased sensitivity to bacteriophage P1 infection). Differential gene expression and gel shift assays demonstrated that YncC is a repressor of the predicted periplasmic protein-encoding gene, ybiM, which was corroborated by the isogenic yncC ybiM double mutation that repressed the yncC phenotypes (biofilm formation, colanic acid overproduction, mucoidy and bacteriophage resistance). Through nickel-enrichment DNA microarrays and additional gel shift assays, we found that the putative transcription factor B3023 (directly upstream of mqsR) binds the yncC promoter. Overexpressing MqsR, AI-2 import regulators LsrR/LsrK and AI-2 exporter TqsA induced yncC transcription, whereas the AI-2 synthase LuxS and B3023 repressed yncC. MqsR has a toxic effect on E. coli bacterial growth, which is partially reduced by the b3023 mutation. Therefore, AI-2 quorum-sensing control of biofilm formation is mediated through regulator MqsR that induces expression of the transcription factor YncC. YncC inhibits the expression of periplasmic YbiM, which prevents overproduction of colanic acid (excess colanic acid causes mucoidy) and prevents YbiM from inhibiting biofilm formation.

RNA interference (RNAi) is an evolutionarily conserved process of gene silencing in multiple organisms, which has become a powerful tool for investigating gene function by reverse genetics. Recently, many groups have reported to use synthesized oligonucleotides or siRNA encoding plasmids to induce RNAi in mammalian cells by transfection, but this is still limited in its application, especially when it is necessary to generate long-term gene silencing in vivo. To circumvent this problem, retrovirus- or lentivirus-delivered RNAi has been developed. Here, we described two retroviral systems for delivering short hairpin RNA (shRNA) transcribed from the H1 promoter. The results showed that retroviral vector-mediated RNAi can substantially downregulate the expression of human p53 in 293-T cells. Furthermore, the retroviral vector-mediated RNAi in our transduction system can stably inactivate the p53 gene for a long time. Compared to shRNAs transcribed from the U6 promoter, H1-driven shRNA also dramatically reduced the expression of p53. The p53 downregulation efficiencies of H1- and U6-driven shRNAs were almost identical. The results indicate that retroviral vector-delivered RNAi would be a useful tool in functional genomics and gene therapy.

The T42 peptide, generated from two active fragments of tumstatin, has been shown to have anti‑tumor activity. The adenoviral vector is the most frequently used vector in research and clinical trials for gene therapy. In the present study, the anti‑tumor activity of the T42 peptide and quadruple T42 (4xT42) peptide adenoviral vectors were elucidated for the first time, to the best of our knowledge. Human embryonic kidney 293 cells were infected with plasmid adenovirus (pAd)‑enhanced green fluorescent protein (EGFP)‑T42 or pAd‑EGFP‑4xT42 and the expression of the T42 and 4xT42 genes was confirmed by the identification of GFP expression and reverse transcription polymerase chain reaction experiments. The anti‑cancer effects of pAd‑EGFP‑T42 and pAd‑EGFP‑4xT42 on breast cancer cells in vivo and in vitro were subsequently investigated. The results indicated that the packaging of the recombinant adenoviruses with the viral titer was successful, following purification at 5x109 plaque forming units/ml. The results also revealed that the recombinant adenoviruses promoted apoptosis in MCF‑7 breast cancer cells and inhibited cancer growth. Through the analysis of caspase‑3, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein expression, it was demonstrated that the T42/4xT42 peptide may induce apoptosis via the mitochondrial pathway. In addition, mouse xenograft experiments confirmed that the T42 peptide inhibited tumor growth and reduced angiogenesis in vivo. In conclusion, the results of the present study indicated that the T42 and 4xT42 peptide genes, transfected by a recombinant adenovirus, may provide a potential novel strategy for the treatment of breast cancer.

To construct the lentiviral vector containing SOX9 gene and to detect its expression in MSCs derived from rabbit bone marrow.Human sox-9 gene coding region fragment was obtained by RT-PCR (reverse transcription-polymerase chain reaction) and then cloned into the plasmid of Pwpxl-MOD2 to form Pwpxl-MOD2/SOX9. Pwpxl-MOD2/SOX9, pRsv-REV, pMDlg-Prre and pMD2G were co-transfected into 293T cells to obtain recombinant virus containing SOX9 gene. Meanwhile, Pwpxl-MOD2, pRsv-REV, pMDlg-pRRE and pMD2G were transfected into another group of 293T cells as a control group packing into blank lentiviral vector. Then the packed lentiviral vector was transfected into MSCs which derived from rabbit bone marrow. The expression of SOX9 was detected by both RT-PCR and Western blot. Identification and proliferation of MSCs was determined by MTT after transfection.The sequencing and restriction analysis showed that SOX9 gene fragment was correctly connected and cloned into the plasmid Pwpxl-MOD in lentiviral vectors. After transfection, the expression of SOX9 gene in MSCs was confirmed by RT-PCR and Western blot. MTT showed the growth of MSCs had no significant effect after transfection with lentiviral vector.Lentiviral vector carrying SOX9 gene has been successfully constructed. There is a stable expression in transfected MSCs. Thus it will facilitate the exploratory development of gene and biological therapy for intervertebral disc degeneration.

Mammalian cells express several distinct isoforms of transcription factor CREB (cAMP-responsive element binding protein). At least two forms, alpha- and delta CREB, arise through alternative splicing of the CREB gene transcript. In this communication we demonstrate that the mRNAs of several CREB isoforms are expressed in rat C6 glioma cells and that the intracellular levels of these mRNAs are markedly induced by the synthetic glucocorticoid dexamethasone. Nuclear run-off assays show that the induction occurs, at least in part, through a transcriptional mechanism. The enhanced cellular levels of CREB mRNAs are accompanied by increased CREB protein and CRE-binding activity of nuclear extracts as evaluated by immunoblot and Southwestern blot assays.

C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. The aim of the present study is to investigate the effects of CRP on the production of adiponectin in 3T3-L1 adipocytes. Northern and western blot analysis revealed that CRP treatment inhibited adiponectin mRNA expression and secretion in a dose- and time-dependent manner. Co-incubation of adipocytes with rosiglitazone and CRP decreased induction of adiponectin gene expression by rosiglitazone. However, luciferase reporter assays did not show that CRP affected the activity of ~2.1 kb adiponectin gene promoter, which was increased by rosiglitazone alone. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by LY294002 partially reversed inhibition of adiponectin gene expression by CRP. These results collectively suggest that CRP suppresses adiponectin gene expression partially through the PI-3 kinase pathway, and that decreased production of adiponectin might represent a mechanism by which CRP regulates insulin sensitivity.

Background Development in higher eukaryotes involves programmed gene expression. Cell type-specific gene expression is established during this process and is inherited in succeeding cell cycles. Higher eukaryotes have evolved elegant mechanisms by which committed gene-expression states are transmitted through numerous cell divisions. Previous studies have shown that both DNase I-sensitive sites and the basal transcription factor TFIID remain on silenced mitotic chromosomes, suggesting that certain trans-factors might act as bookmarks, maintaining the information and transmitting it to the next generation. Results We used the mouse globin gene clusters as a model system to examine the retention of active information on M-phase chromosomes and its contribution to the persistence of transcriptional competence of these gene clusters in murine erythroleukemia cells. In cells arrested in mitosis, the erythroid-specific activator NF-E2p45 remained associated with its binding sites on the globin gene loci, while the other major erythroid factor, GATA-1, was removed from chromosome. Moreover, despite mitotic chromatin condensation, the distant regulatory regions and promoters of transcriptionally competent globin gene loci are marked by a preserved histone code consisting in active histone modifications such as H3 acetylation, H3-K4 dimethylation and K79 dimethylation. Further analysis showed that other active genes are also locally marked by the preserved active histone code throughout mitotic inactivation of transcription. Conclusion Our results imply that certain kinds of specific protein factors and active histone modifications function as cellular memory markers for both competent and active genes during mitosis, and serve as a reactivated core for the resumption of transcription when the cells exit mitosis.

To investigate the expression of early growth response gene-1 (Egr-1) and its correlative genes: platelet-derived growth factor-B (PDGF-B) gene and transforming growth factor-beta(1) (TGF-beta(1)) gene, in autogenous vein graft and the relationship of the expression of these genes to intimal hyperplasia (IH).A segment of the right common jugular vein was transplanted to the infra-renal abdominal aorta of the same individual so as to establish an autogenous vein graft model in 90 Wistar rats. Vein samples were harvested 1, 2, 6, and 24 hours, 3 and 7 days, and 2, 4, and 6 weeks after the operation to undergo histological examination. In situ hybridization and RT-OCR were performed to examine the mRNA expression of Egr-1, PDGF-B, and TGF-beta(1). Western blotting and immunohistochemistry were used to detect the protein expression of Egr-1, PDGF-B, and TGF-beta(1). The contralateral common jugular vein was used as control.Histological examination showed a great amount of vascular smooth muscle cells (VSMCs) in the tunica intima and tunica media of the vein graft since the 7 th day after the transplantation. mRNA expression of Egr-1, PDGF-B, and TGF-beta(1) was not found in the normal veins. The expression of Egr-1 mRNA in the vein graft showed a biphasic manner: increased remarkably 1 h after the transplantation, decreased during the period 6 h approximately 3 d after, re-increased 7 d after, and peaked 4 weeks after. The mRNA expression of PDGF-B appeared 6 h after, and peaked 2 weeks after, and began to decrease since the 4 th week. The mRNA expression of TGF-beta(1) increased 6 h after, peaked 7d after, and began to decrease 2 weeks after. The protein expression of Egr-1 appeared 2 h after, and peaked 4 weeks after; the protein expression of PDGF-B appeared 6 h after and peaked 4 weeks after; and the protein expression of TGF-beta(1). Appeared 24 h after and peaked 2 weeks after. Immunohistochemistry showed that Egr-1 protein expression was mainly in the VSMCs and monocytes/macrophages in the tunica media in the early stage after transplantation, and peaked 4 weeks later with a positive cell rate of 40% +/- 9%; in the early stage after transplantation PDGF-B protein expression was mainly in the VSMCs in the tunica media, and peaked 4 weeks after with a positive cell rate of 45% +/- 4%; the TGF-beta(1) protein expression peaked 2 weeks after with a positive cell rate of 41% +/- 7%; and 4 weeks after transplantation the protein expressions of Egr-1, PDGF-B, and TGF-beta(1) were all mainly in the VSMCs in the newly-grown tunica intima.The IH of vein graft is affinitive with the expression of Egr-1, PDGF-B, and TGF-beta(1). The activation and expression of Egr-1, PDGF-B, and TGF-beta(1) depend on Egr-1 and may contribute to the high expression of Egr1 via a feedback mechanism.

To investigate the anti-virus infection activity of internal ribosome entry site (IRES) specific inhibitor RNA (IRNA).IRNA eukaryotic vector pcRz-IRNA or mIRNA eukaryotic vector pcRz-mIRNA was transfected into human hepatocarcinoma cells (HHCC), then selected with neomycin G418 for 4 to 8 weeks, and then infected with polio virus vaccines line. The cytopethogenesis effect was investigated and the cell extract was collected. At last the polio virus titer of different cells was determined by plaque assay.Constructive expression of IRNA was not detrimental to cell growth. HCV IRES-mediated cap-independent translation was markedly inhibited in cells constructively expressing IRNA compared to control hepatoma cells. However, cap-dependent translation was not significantly affected in these cell line. Additionally, HHCC cells constitutively expressing IRNA became refractory to infection of polio virus.IRES specific IRNA can inhibit HCV IRES mediated translation and poliovirus replication.

Acute ocular hypertension (AOH) is a condition found in acute glaucoma. The purpose of this study is to investigate the protective effect of Lycium barbarum polysaccharides (LBP) and its protective mechanisms in the AOH insult. LBP has been shown to exhibit neuroprotective effect in the chronic ocular hypertension (COH) experiments. AOH mouse model was induced in unilateral eye for one hour by introducing 90 mmHg ocular pressure. The animal was fed with LBP solution (1 mg/kg) or vehicle daily from 7 days before the AOH insult till sacrifice at either day 4 or day 7 post insult. The neuroprotective effects of LBP on retinal ganglion cells (RGCs) and blood-retinal-barrier (BRB) were evaluated. In control AOH retina, loss of RGCs, thinning of IRL thickness, increased IgG leakage, broken tight junctions, and decreased density of retinal blood vessels were observed. However, in LBP-treated AOH retina, there was less loss of RGCs with thinning of IRL thickness, IgG leakage, more continued structure of tight junctions associated with higher level of occludin protein and the recovery of the blood vessel density when compared with vehicle-treated AOH retina. Moreover, we found that LBP provides neuroprotection by down-regulating RAGE, ET-1, Aβ and AGE in the retina, as well as their related signaling pathways, which was related to inhibiting vascular damages and the neuronal degeneration in AOH insults. The present study suggests that LBP could prevent damage to RGCs from AOH-induced ischemic injury; furthermore, through its effects on blood vessel protection, LBP would also be a potential treatment for vascular-related retinopathy.

Background Mucin alterations are a common feature of esophageal neoplasia, and alterations in MUC2 mucin have been associated with tumor progression in the esophagus. Bile acids have been linked to esophageal adenocarcinoma and mucin secretion, but their effects on mucin gene expression in human esophageal adenocarcinoma cells is unknown. Methods Human esophageal adenocarcinoma cells were treated 18 hours with 50–300 μM deoxycholic acid, chenodeoxycholic acid, or taurocholic acid. MUC2 transcription was assayed using a MUC2 promoter reporter luciferase construct and MUC2 protein was assayed by Western blot analysis. Transcription Nuclear factor-κB activity was measured using a Nuclear factor-κB reporter construct and confirmed by Western blot analysis for Nuclear factor-κB p65. Results MUC2 transcription and MUC2 protein expression were increased four to five fold by bile acids in a time and dose-dependent manner with no effect on cell viability. Nuclear factor-κB activity was also increased. Treatment with the putative chemopreventive agent aspirin, which decreased Nuclear factor-κB activity, also decreased MUC2 transcription. Nuclear factor-κB p65 siRNA decreased MUC2 transcription, confirming the significance of Nuclear factor-κB in MUC2 induction by deoxycholic acid. Calphostin C, a specific inhibitor of protein kinase C (PKC), greatly decreased bile acid induced MUC2 transcription and Nuclear factor-κB activity, whereas inhibitors of MAP kinase had no effect. Conclusion Deoxycholic acid induced MUC2 overexpression in human esophageal adenocarcinoma cells by activation of Nuclear factor-κB transcription through a process involving PKC-dependent but not PKA, independent of activation of MAP kinase.

AIM: To investigate the inhibitory effect of hepatitis C virus internal ribosome entry site (HCV IRES) specific inhibitor RNA (IRNA) on gene expression mediated by HCV IRES in vivo. METHODS: By using G418 screening system, hepatoma cells constitutively expressing IRNA or mutant IRNA (mIRNA) were established and characterized, and HCV replicons containing the 5’ untranslated region (5’UTR) were constructed by using the same method. Cotransfection of pCMVNCRluc containing HCV 5’UTR-luc fusion genes and eukaryotic vector of IRNA into human hepatic carcinoma cells (HepG2) was performed and the eukaryotic expression plasmid of IRNA was transfected transiently into HCV replicons. pCMVNCRluc or pCDNA-luc was cotransfected with pSV40-β Gal into IRNA expressing hepatoma cells by using lipofectamine 2000 in vitro. Then the reporting gene expression level was examined at 48 h after transfection by using a luminometer and the expressing level of HCV C antigen was analysed with a confocal microscope. RESULTS: Transient expression of IRES specific IRNA could significantly inhibit the expression of reporter gene and viral antigen mediated by HCV IRES by 50% to 90% in vivo, but mIRNA lost its inhibitory activity completely. The luciferase gene expression mediated by HCV IRES was blocked in the HHCC constitutively expressing IRNA. At 48 h after transfection, the expression level of reportor gene descreased by 20%, but cap-dependent luciferase gene expression was not affected. IRNA could inhibit the HCV replicon expression 24 h after transfection and the highest inhibitory activity was 80% by 72 h, and the inhibitory activity was not increased until 7d after transfection. CONCLUSION: IRNA can inhibit HCV IRES mediated gene expression in vivo.

AIM: To confirm the existence of CD226 ligand and its distribution, which is a novel molecule that was cloned in 1996. METHODS: The mRNA was extracted from TPA activated Jurkat cells and used as a template for reverse-transcription. After PCR amplification, the fragment including CD226 extracellular region and the splice donor sequence “ACTTACCTGT” was obtained and cloned into fusion expression vector pIG. The recombinant vector pCD226/Ig was transfected in COS-7 cells by DEAE-Dextran method, the secreting fusion protein was identified by Sandwich ELISA, and was purified by anti-CD226 affinity chromatography. This fusion protein was used as a probe in the investigation of CD226 ligand by immunohistochemistry. Existence of CD226 ligand was further identified by adhesion experiment. RESULTS: Expression of a secreting fusion protein was identified by sandwich ELISA, indicating that both CD226 extracellular domain and IgGFc domain could be recognized respectively by anti-CD226 and anti-hIgFc mAb. About 130 μg CD226/Ig fusion protein could be obtained from 100 mL COS-7 culture supernatants by anti-CD226 affinity chromatography purification. SDS-PAGE showed that this fusion protein has a molecular mass of 83 ku. It was confirmed by immunohistochemistry that CD226 ligand expressed on the Colo205 cells, but not on Jurkat cell, U937 cell and mixed lymphocyte culture cells. In adhesive assay, resting Jurkat cells did not have significant adhesion to Colo205 cells. In contrast, activated Jurkat cells could bind to colon carcinoma Colo205 cells and this adhesive reaction could be blocked by CD226/Ig fusion protein or anti-CD226 mAb. Immunochemical experiment showed that Colo205 cells could be specifically stained by CD226/Ig, indicating that CD226 ligand exists on the surface of Colo205 cells. CONCLUSION: Existence of CD226 ligand on the surface of Colo205 cells was identified by immunohistochemistry and adhesion blocking experiment. In addition, the secreting CD226/Ig fusion protein prepared in this study will be a potential tool for further investigation of CD226 ligand.