Microgram Of Total RNA Research Articles

Analysis of Queuosine tRNA Modification Using APB Northern Blot Assay.

Queuosine (Q) is a hypermodified base that occurs at the wobble position of transfer RNAs (tRNAs) with a GUN anticodon. Q-tRNA modification is widespread among eukaryotes, yet bacteria are the original source of Q. Eukaryotes acquire Q from their diet, or from the gut microbiota (in multicellular organisms). Despite decades of study, the detailed roles of Q-tRNA modification remain to be elucidated, especially regarding its specific mechanisms of action. Here, we describe a method for the fast and reliable detection of Q-tRNA modification levels in individual tRNAs using a few micrograms of total RNA as starting material. The methodology is based on the co-polymerization of boronic acid (N-acryloyl-3-aminophenylboronic acid (APB)) in polyacrylamide gels, and on the interplay between this derivative and free cis-diol groups of the tRNA. During electrophoresis, the cis-diol groups slow down the Q-modified tRNA, which then can be separated from unmodified tRNA and quantified using Northern blot analysis.

MicroRNA Sequencing of Discoid Lupus Erythematosus Skin

BackgroundDiscoid lupus erythematous (DLE) is an autoimmune skin condition that causes profound disfigurement. Environmental effects are linked to increased risk and worsening disease off activity in DLE. MicroRNA (miRNA) are increasingly found to have important roles in the environmental modulation of inflammation and cellular differentiation in autoimmune diseases.ObjectiveThe goal of the pilot study was to comprehensively identify the miRNA transcriptome in DLE.MethodsThe specimens were from African American women (DLE=4, normal=4) and were age‐matched [median age=55 years (range=50–61), median DLE duration=18 years]. Subjects with DLE did not have concomitant systemic lupus and were not on systemic immunosuppression during skin biopsy. Skin specimens were composed of keratinocytes, epidermal appendages, vasculature, dermal fibroblasts and skin homing immune cell populations. We extracted total RNA using a method that preserved small RNAs (Ambion). RNA was prepared for sequencing with the Small RNA Sample Prep Kit,□□ □□ adapters were ligated to approximately 1 microgram of total RNA. Each library was loaded on a single Illumina lane at 20 picogram and subjected to 86 cycles of sequencing on HiSeq. We generated 52.4 million raw and 15.3 million qualified reads. We mapped qualified reads to miRNA precursors, functional non‐coding RNAs and build 37 (hg19) of the human genome. The adapter‐trimmed, qualified reads were distributed around an average length of 21 nucleotides. The number of reads and mapping proportions in the DLE group approximate that in the normal skin groups. We detected mature miRNAs derived from 1240 known miRNA precursor families. These were represented by at least one read in the cumulative data set. We analyzed the data from our next generation sequencing based on relative comparisons of normalized read counts between DLE and normal skin to detect relative changes. Standardized fold changes of miRNAs were calculated as mean differences in read counts divided by pooled standard deviations in the log scale.ResultsWe identified 18 known miRNAs that were significantly over or under expressed in active DLE lesions versus normal skin (p‐values < 0.05). Among these are mir‐146a, mir‐223, mir‐21, mir‐23b. These miRNAs are linked to IFN‐regulation, hypomethylation, ultraviolet exposure, differentiation of the keratinocytes, epidermal dendritic cells, and T cells, and estrogen response.ConclusionFrom this discovery‐based study, we have identified several miRNAs that may play an important role in epigenetic regulation in DLE that ultimately affects disease severity.Support or Funding InformationCTSA of University of CincinnatiThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of SOCS1 and SOCS3 Genes in Interferon-Treated and Direct-Acting Antiviral Drugs-Treated Hepatitis C Patients.

Genetics of host plays a significant role in susceptibility and pathogenesis of disease. During hepatitis C virus (HCV) infection, HCV proteins interfere with interferon (IFN) signaling pathways and upregulate transcription of suppressor of cytokine signaling 1 and 3 genes (SOCS1 and SOCS3), which results in impaired immune response. In this study, we evaluated relative expression of SOCS1 and SOCS3 in untreated HCV patients and patients treated with 2 different treatment strategies that are, (IFN therapy and direct-acting antiviral (DAA) drug regimen. To study gene expression, peripheral blood mononuclear cells (PBMCs) were isolated by using Histopaque. Total RNA was extracted from PBMCs by using BIOzol. Nine microgram of total RNA from each sample was used and reverse transcribed into single-stranded complementary DNA (cDNA) by using M-MLV reverse transcriptase (Invitrogen). The synthesized cDNA was diluted to a final concentration of 500 ng/μL. This diluted cDNA was further used for expression analysis of SOCS1and SOCS3 genes using Rotor Gene Q Real-Time PCR Detection System (QIAGEN). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a housekeeping gene. We found that the SOCS1 expression in IFN and DAA-treated patient groups was 5.4 fold and 1.2 fold, respectively, high compared with the healthy controls (IFN versus healthy, P = 0.019 and DAA versus healthy, P = 0.91), whereas the SOCS3 expression in IFN and DAA-treated patient groups was 3.7 fold and 2 fold, respectively, high in comparison with the expression in healthy controls (IFN versus healthy, P = 0.025 and DAA versus healthy, P = 0.03). We also found a significant difference in the relative expression of SOCS1 and SOCS3 in DAAs-treated and IFN/ribavirin (RBV)-treated and untreated individual. We concluded that by targeting HCV proteins with DAAs, SOCS1, and SOCS3 transcription can be more effectively normalized compared to the treatment with IFN/RBV therapy.

Comparison of MRD Levels and Gene Expression Patterns in MLL-R Versus Non-MLL Infant ALL

Abstract Introduction Acute lymphoblastic leukaemia (ALL) in infants has poor overall survival despite being characterized by very few genetic aberrations per case. The most common genetic change, present in over 75% of cases, is the rearrangement of the mixed lineage leukaemia (MLL/KMT2A) gene (MLL-R) that also occurs in AML and mixed phenotype acute leukaemia (MPAL). Because infant ALL is rare and distinctive, most Australian and New Zealand patients are enrolled on specific clinical trials. In earlier infant ALL trials, MRD was not used for risk stratification firstly because of the difficulty of finding sensitive markers for specific immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements and the secondly because, in infant ALL, Ig/TCR markers are often present in sub-clones that can be lost at relapse due to clonal selection. MRD testing is performed in the current Interfant 06 trial preferentially using markers based on the genomic breakpoint sequences of MLL gene rearrangements. The treatment of infant ALL remains very challenging with relatively poor survival rates attributable to both toxicity and relapse. The objectives of this study were therefore to analyse MRD data for Interfant 06 patients enrolled at ANZCHOG centres and to perform a pilot experiment evaluating gene expression for key genes in infant ALL samples on a microfluidics platform, as a basis for identifying potential targeted therapies. Methods MRD was measured in bone marrow DNA from Interfant 06 patients enrolled since 2006, using sensitive Real-time Quantitative PCR (PCR-MRD) patient-specific assays to detect either MLL gene rearrangements (in 17) and/or conventional immunoglobulin and T-cell receptor (Ig/TCR) markers (21). Gene expression levels for 90 genes important in childhood cancers were measured by Taqman-based microfluidic assays in duplicate using cDNA from 2 micrograms of total RNA from 10 infant ALL samples (5 MLL, 5 non-MLL) at diagnosis (6) or relapse (4). Results Patient-specific PCR-MRD tests were developed for 27 out of 28 Australian and New Zealand infant ALL patients. 17/18 MLL-R ALL patients had MLL-R assays and 21/28 patients had Ig/TCR MRD tests, with only 1 (non-MLL) patient having no MRD markers. There was a wide range of MRD responses to induction therapy (Figure 1). Bone marrow MRD at the end of induction was high (>1x10-3) in 44% of MLL-R ALL infants compared to 25% of non-MLL ALL infants and 15% of older children enrolled on ANZCHOG ALL8. In 8/13 MLL-R patients who had both types of marker, MRD levels were higher when measured by their MLL-R marker than by their Ig/TCR marker. In a set of 90 genes selected for expression analysis, higher levels were found for 17 genes in 2 or more of the 10 infant ALL samples evaluated. These more highly expressed genes included potential or known drug targets BCL2, ERBB2, ERBB4, ILRA2, CSF1R and PARP1. Conclusions The quantitation of MRD based on MLL rearrangements in ALL is effective and can also be used to monitor response to therapy in infant ALL as well as MLL-R cases of AML and MPAL. The combined application of MLL-R and Ig/TCR markers allowed 97% of infant ALL patients to be MRD monitored with a sensitive marker. In most patients with both type of MRD marker, higher levels of MRD were detected in end of induction samples using the MLL versus Ig/TCR tests. One interpretation is that Ig/TCR genes are rearranged after the MLL rearrangement in ALL sub-clones that are both more mature and more chemo-sensitive. This finding also confirms the current consensus that disease-related MLL-R markers provide better risk assessment than Ig/TCR markers. Our Fluidigm analysis has shown that quantitative measurement of multiple gene expressions is feasible on small RNA samples and can be used to rapidly screen for specific expression of genes coding for drug targets in ALL patients. Support: NHMRC Australia APP1057746, Sporting Chance Cancer Foundation. Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Figure 1. Comparison of MRD response to induction therapy in MLL-R infant ALL compared with non-MLL infant ALL (Interfant 06) and older children (ANZCHOG ALL8). Disclosures No relevant conflicts of interest to declare.

A Novel Multi-Gene Array Allows Relapse Risk Stratification in Acute Myeloid Leukemia Patients Undergoing Stem Cell Transplantation

Background:Acute myeloid leukemia (AML) is a diagnosis encompassing a diverse group of myeloid malignancies. Heterogeneous genetic etiology, together with the potential for oligoclonality within the individual patient, has made the identification of a single high sensitivity marker of disease burden challenging. PCR based assessment of WT1 expression has been extensively tested as a marker of submicroscopic AML disease burden but is not universally overexpressed in all cases. Our hypothesis was that the addition of other individually suboptimal gene expression assays to the backbone of WT1 PCR based testing in a multi-gene array format would improve prediction of relapse and survival when tested prior to allogeneic stem cell transplantation (allo-SCT).Patients and Methods:We identified pre-transplantation peripheral blood samples from 74 AML patients who had received allo-SCT as part of NHLBI clinical protocols performed at the NIH clinical center in Bethesda, Maryland between 1994 and 2012. All had morphological evaluation of disease status within the two months prior to transplantation and at least twelve months of documented post-SCT clinical outcomes data or until date of death if this occurred before twelve months. Transplantswere predominately myeloablative, T cell depleted, peripheral blood stem cell sibling matched allogeneic transplants with cyclosporine-based graft versus host disease prophylaxis. Peripheral blood samples from fifty healthy adults donors were used as baseline controls. All clinical protocols and permission to use blood for research were conducted in accordance with Declaration of Helsinki principles and were approved by the Institutional Review Board with written informed consent obtained from all subjects.One microgram of total RNA isolated from cryostored peripheral blood samples was reverse-transcribed into cDNA and used for quantitative real time PCR (qRT-PCR) reactions using custom multi-gene PCR array plates (MG-MRD) and SYBR green, with normalization to c-abl. Arrays included primers for detection of WT1, CCNA1, PRTN3, PRAME, MSLN and ABL transcripts. Thresholds for positivity were established based on expression levels seen in healthy normal donors. Statistical analysis was performed using GraphPad Prism (La Jolla, CA, USA). Comparison between survival or relapse curves was performed using the Log-rank (Mantel-Cox) test.Results:In this cohort of 74 patients the overall survival observed at three years after transplant (3yr OS) was calculated to be 36%. Traditional response criteria from examination of bone marrow risk stratified these 74 patients into two groups, the morphological CR group (n=48) with a 3yr OS of 48% and a group of 26 patents with active (refractory/refractory) disease at the time of allo-SCT with a 3yr OS of only 15%. Peripheral blood testing for WT1 overexpression identified 25 patients as positive (3 of whom had no clinically evident disease by morphology on bone marrow examination, ie; minimal residual disease, MRD). Testing using the MG-MRD assay identified an additional 9 patients from the WT1 negative group as also having residual disease (2 had morphologically evident disease, 7 with MRD). The mortality in this group of patients following transplantation was 100%. The 34 (of the total 74) patients with positive MG-MRD prior to transplantation had a three year overall survival of just 9%.This MG-MRD positive group also experienced 22 out of a total of 24 relapses observed in the first year following transplantation. Pre-SCT MG-MRD testing correctly predicted all cases of early relapse (ie: within 100 days after allo-SCT) compared to just 57% (8/14) using WT1 alone. For the 37 patients who entered transplant in a pathological CR, and who did not suffer non-relapse mortality in the first year, the 1yr relapse rate was 24%. Pre-SCT WT1 testing had 100% positive predictive value (PPV) but only 33% sensitivity in identifying relapses in this subgroup, compared to 100% PPV and 89% sensitivity for the MG-MRD array.Conclusion:PCR based pre-SCT MRD testing using a multi-gene panel outperformed WT1 alone, correctly identifying 36% more patients (n=9, all of whom died) as belonging to a group at high-risk for post-SCT relapse and death. Collaborations are now being sought to validate these findings in additional cohorts. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Notch Transcriptional Control of Vascular Smooth Muscle Regulatory Gene Expression and Function

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

Molecular Markers of Spermatogonial Stem Cells in the Domestic Cat

Spermatogonial stem cells (SSCs) represent an exciting new avenue for assisted reproduction in endangered and genetically valuable species. Before this technology can be applied to wildlife, species-specific markers are required to evaluate SSC enrichment strategies and monitor subsequent in vitro culture. This study was designed to evaluate six conserved SSC markers (THY1, GPR125, GFRalpha1, PLZF, UCHL1 and OCT4) in the cat. Testes from three juveniles and three adults were obtained following routine castrations and processed for mRNA extraction. RT-PCR of whole testis and cell suspensions enriched for SSCs by differential plating confirmed that all six SSC markers are expressed in both the whole testis and SSC-enriched cell fractions. The expression of all six putative SSC marker genes in the cat testis suggests conservation of SSC markers, and perhaps self-renewal mechanisms, in felids.

1,25-Dihydroxyvitamin D3 Suppresses Telomerase Expression and Human Cancer Growth through MicroRNA-498

Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).

Age‐ and Ethanol Concentration‐Dependent Effects of Acute Binge Drinking in the HIV‐1 Transgenic Rat

Binge drinking is common in young people. Alcoholic beverages vary significantly in their ethanol (EtOH) concentration (alcohol by volume). We previously showed EtOH concentration-dependent activation of the hypothalamic supraoptic nucleus. In the HIV-infected population, incidence of alcohol abuse is close to 50%. We found age-dependent expression of HIV-1 viral proteins in the HIV-1 transgenic (HIV-1Tg) rat. Thus, we hypothesized that there are age- and EtOH concentration-dependent effects of binge drinking in HIV-1-positive individuals. Blood ethanol concentration was measured in adult F344 rats after gavage (i.g.) administration of water, 20% EtOH, or 52% EtOH. We also compared expression of the HIV-1 viral protein Tat in the brain, spleen, and liver of adult and adolescent HIV-1Tg rats following binge i.g. administration of water, 20% EtOH, or 52% EtOH for 3 days (4.8 g/kg/d) using absolute quantitative real-time reverse transcription-polymerase chain reaction. In a parallel study, we assessed age-dependent motor function in the HIV-1Tg rats 1 day after exposure to 20% EtOH using the open-field test. Blood ethanol concentration was significantly higher in the 52% EtOH-treated F344 rats compared to the 20% EtOH animals at 90 minutes posttreatment. In the adult HIV-1Tg rats, HIV-1 Tat expression (copies per microgram of total RNA) was significantly increased in the brain, liver, and spleen of the 52% EtOH group, but not in the 20% EtOH group. However, in the adolescent animals, HIV-1 Tat expression in the 52% EtOH group was increased in the brain and liver, but not in the spleen. A significant reduction in locomotor activity occurred in 20% EtOH-treated adult HIV-1Tg rats compared to the water control, although no difference was observed in the adolescent HIV-1Tg animals. Our data indicate that binge alcohol drinking can have age- and EtOH concentration-dependent effects in the presence of HIV-1 infection.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

Aberrant Stratifin Overexpression Is Regulated by Tumor-Associated CpG Demethylation in Lung Adenocarcinoma

We previously have shown the aberrant overexpression of stratifin (SFN, 14-3-3 ς) in lung adenocarcinoma. Although SFN is known to facilitate tumor cell proliferation, the mechanism that underlies its aberrant expression has remained unclear. SFN, the downstream target of p53, often has been reported to be hypermethylated and subsequently silenced in certain cancers; however, its hypomethylation-linked reactivation has not yet been validated. In this study, we investigated the DNA methylation status of the SFN promoter region using 8 lung cancer cell lines and 32 specimens of adenocarcinoma tissue. Real-time methylation-specific PCR analysis showed that although both normal lung tissue and adenocarcinoma in situ bore a completely methylated SFN promoter, the promoter region in almost all invasive adenocarcinomas was at least partially methylated. The expression of SFN and its level of methylation were correlated strongly. Furthermore, statistical analysis revealed that the level of methylation became reduced with progression of the pathologic stage, although no clear relationship between methylation level and p53 abnormality was found. These results suggest that methylation-related silencing of SFN occurs in both normal lung tissues and adenocarcinoma in situ, and that demethylation of the SFN promoter participates in the aberrant expression of SFN in invasive adenocarcinoma cells, independently of p53 alteration. This novel finding might be informative for clarifying the mechanism that underlies the progression of early lung adenocarcinoma.

Production of large numbers of plasmacytoid dendritic cells with functional activities from CD34+ hematopoietic progenitor cells: Use of interleukin-3

Plasmacytoid dendritic cells (pDC), a subset of dendritic cells characterized by a rapid and massive type-I interferon secretion through the Toll-like receptor pathway in response to viral infection, play important roles in the pathogenesis of several diseases, such as chronic viral infections (e.g., hepatitis C virus, human immunodeficiency virus), autoimmunity (e.g., psoriasis, systemic lupus erythematosus), and cancer. As pDC represent a rare cell type in the peripheral blood, the goal of this study was to develop a new method to efficiently generate large numbers of cells from a limited number of CD34(+) cord blood progenitors to provide a tool to resolve important questions about how pDC mediate tolerance, autoimmunity, and cancer. Human CD34(+) hematopoietic progenitor cells isolated from cord blood were cultured with a combination of Flt3-ligand (Flt3L), thrombopoietin (TPO), and one of the following cytokine: interleukin (IL)-3, interferon-β(IFN-β), or prostaglandin E2(PGE(2)). Cells obtained in the different culture conditions were analyzed for their phenotype and functional characteristics. The addition of IL-3 cooperates with Flt3L and TPO in the induction of pDC from CD34(+) hematopoietic progenitor cells. Indeed, Flt3L/TPO alone or supplemented with prostaglandin E2 or interferon-β produced smaller amounts of pDC from hematopoietic progenitor cells. In addition, pDC generated in Flt3L/TPO/IL-3 cultures exhibited morphological, immunohistochemical, and functional features of peripheral blood pDC. We showed that IL-3, in association with Flt3L and TPO, provides an advantageous tool for large-scale generation of pDC. This culture condition generated, starting from 2 × 10(5) CD34(+) cells, up to 2.6 × 10(6) pDC presenting features of blood pDC.

Differential Gene Expression Profile of the Mouse Oviduct During the Estrous Cycle.

The mammalian oviduct provides suitable environment for gamete's transport, interaction, fertilization and early embryo development. Gamete's arrival in the oviduct may cause distinct alterations in the oviductal gene expression. So far, there are only few studies providing the limited information regarding the oviductal gene expression profile. It is thus still not enough for better understanding of oviductal physiology. In this study, we attempted to reveal the profile of the oviductal transcriptome at each stage of estrous cycle by microarray analyses. The oviductal tissue was separately collected at four stage of the estrous cycle, i.e., proestrus, estrus, metestrus, and diestrus. The estrous cycle was staged by examining vaginal smears. One microgram of total RNA was transcribed into cDNA, reversed transcribed via in vitro transcription, and labeled with biotin. The bead-based microarray (Illumina BeadArray) system was used to profile the oviductal transcriptome at each stage of the estrous cycle. Microarray data were validated by quantitative reverse transcription-PCR (real-time PCR) measurements. Cluster analysis of microarray data was performed using Genesis software. Ingenuity Pathways Analysis (IPA) software was used for finding function and pathway for specific biological states. Our results showed that differentially expressed genes, 199, 131, and 13, genes with a 1.5-fold difference were observed in the oviduct at proestrus, estrus, and metestrus, respectively, as compared individually with the diestrus stage. Ten randomly selected genes were selected and demonstrated to have the same expression trend between the microarray and real-time PCR analysis. These differentially expressed genes could be classified into 5 clusters according the expression pattern. Gene interaction analysis using IPA tool revealed at least 10 associated network functions. Among them, lipid metabolism, development and function of reproductive system, and endocrine system are the most associated network functions. This is the first study to provide a comprehensive gene expression profile of oviduct at the physiological estrus cycle. It may also provide information for our better understanding the complexity of the oviductal biology in creating a hospital environment for gametes interaction. (poster)

GnRH agonist (buserelin)-induced in vitro apoptosis in bovine endometrium

Singh, R., Pretheeban, T. and Rajamahendran, R. 2011. GnRH agonist (buserelin)-induced in vitro apoptosis in bovine endometrium. Can. J. Anim. Sci. 91: 265-273. Apoptosis is a vital physiological process. The local modulatory role of the GnRH, GnRH-R system in uterine physiology is not clear. We investigated GnRH agonist (buserelin)-induced apoptosis in bovine endometrium. Reproductive tracts were collected from a local abattoir. The endometrial explants were sliced into smaller pieces, cultured for 20 h and then treated (6 h) with buserelin (0, 200, 500, 1000 ng mL-1), the GnRH antagonist-antide (500 ng mL-1) and antide buserelin (500 200 ng mL-1), and stored at -80°C for RNA extraction. Two micrograms of total RNA was subjected to reverse transcription-polymerase chain reaction using gene-specific primers. Subsequently, endometrial epithelial cells were isolated from the follicular and luteal phase uteri, cultured for 48 h, characterized and treated with buserelin (200 ng mL-1), antide (500 ng mL-1), and antide buserelin (500 200 ng mL-1) for 6 h. The cells were stained with acridine orange-ethidium bromide and visualized and counted under a fluorescent microscope. Buserelin up-regulated BAX (200 ng mL-1) and CASPASE3 mRNA (200 and 500 ng mL-1) and induced apoptosis (200 ng mL-1) at the cellular level in the follicular phase endometrium. GnRH appears to regulate uterine homeostasis in bovine endometrium at the transcriptional and cellular levels.

GnRH agonist (buserelin)-induced in vitro apoptosis in bovine endometrium

Singh, R., Pretheeban, T. and Rajamahendran, R. 2011. GnRH agonist (buserelin)-induced in vitro apoptosis in bovine endometrium. Can. J. Anim. Sci. 91: 265–273. Apoptosis is a vital physiological process. The local modulatory role of the GnRH, GnRH-R system in uterine physiology is not clear. We investigated GnRH agonist (buserelin)-induced apoptosis in bovine endometrium. Reproductive tracts were collected from a local abattoir. The endometrial explants were sliced into smaller pieces, cultured for 20 h and then treated (6 h) with buserelin (0, 200, 500, 1000 ng mL−1), the GnRH antagonist-antide (500 ng mL−1) and antide+buserelin (500+200 ng mL−1), and stored at −80°C for RNA extraction. Two micrograms of total RNA was subjected to reverse transcription-polymerase chain reaction using gene-specific primers. Subsequently, endometrial epithelial cells were isolated from the follicular and luteal phase uteri, cultured for 48 h, characterized and treated with buserelin (200 ng mL−1), antide (500 ng mL−1), and antide+buserelin (500+200 ng mL−1) for 6 h. The cells were stained with acridine orange-ethidium bromide and visualized and counted under a fluorescent microscope. Buserelin up-regulated BAX (200 ng mL−1) and CASPASE3 mRNA (200 and 500 ng mL−1) and induced apoptosis (200 ng mL−1) at the cellular level in the follicular phase endometrium. GnRH appears to regulate uterine homeostasis in bovine endometrium at the transcriptional and cellular levels.

Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells

TGF-β is a potent inducer of epithelial-to-mesenchymal transition (EMT), a process involved in tumour invasion. TIF1γ participates in TGF-β signalling. To understand the role of TIF1γ in TGF-β signalling and its requirement for EMT, we analysed the TGF-β1 response of human mammary epithelial cell lines. A strong EMT increase was observed in TIF1γ-silenced cells after TGF-β1 treatment, whereas Smad4 inactivation completely blocked this process. Accordingly, the functions of several TIF1γ target genes can be linked to EMT, as shown by microarray analysis. As a negative regulator of Smad4, TIF1γ could be crucial for the regulation of TGF-β signalling. Furthermore, TIF1γ binds to and represses the plasminogen activator inhibitor 1 promoter, demonstrating a direct role of TIF1γ in TGF-β-dependent gene expression. This study shows the molecular relationship between TIF1γ and Smad4 in TGF-β signalling and EMT.

Neuroprotection by Histone Deacetylase-7 (HDAC7) Occurs by Inhibition of c-jun Expression through a Deacetylase-independent Mechanism

Histone deacetylase (HDAC) 7 is a member of the HDAC family of deacetylases. Although some of the HDAC proteins have been shown to regulate neuronal survival and death, whether HDAC7 has a similar role is not known. In this study, we show that HDAC7 protects neurons from apoptosis. In cerebellar granule neurons (CGNs) primed to undergo apoptosis by low potassium treatment, expression of HDAC7 protein is reduced. Reduced expression is also observed in CGNs induced to die by pharmacological inhibition of the proteasome, in cortical neurons treated with homocysteic acid, and in the striatum of R6/2 transgenic mice, a commonly used genetic model of Huntington disease. Forced expression of HDAC7 in cultured CGNs blocks low potassium-induced death, and shRNA-mediated suppression of its expression induces death in otherwise healthy neurons. HDAC7-mediated neuroprotection does not require its catalytic domain and cannot be inhibited by chemical inhibitors of HDACs. Moreover, pharmacological inhibitors of the PI3K-Akt or Raf-MEK-ERK signaling pathways or that of PKA, PKC, and Ca(2+)/calmodulin-dependent protein kinase fail to reduce neuroprotection by HDAC7. We show that stimulation of c-jun expression, an essential feature of neuronal death, is prevented by HDAC7. shRNA-mediated suppression of HDAC7 expression leads to an increase in c-jun expression. Inhibition of c-jun expression by HDAC7 is mediated at the transcriptional level by its direct association with the c-jun gene promoter. Taken together, our results indicate that HDAC7 is a neuroprotective protein acting by a mechanism that is independent of its deacetylase activity but involving the inhibition of c-jun expression.

Identification of Genes Regulated by IL-1β Using Integrative microRNA and mRNA Genomic Analysis in Human Articular Chondrocytes

Objective. The physiological and pathogenetic role of microRNAs (miRNAs) in the maintenance of joint homeostasis and in the development of arthritis is recently being elucidated. In this study, we attempted to identify differentially expressed miRNAs in human osteoarthritis (OA) chondrocytes in response to interleukin (IL)-1β. In addition, simultaneous profiling of miRNA and mRNA expression was performed to get an integrated analysis of miRNA and mRNA expression. Methods. Monolayer cultured chondrocytes obtained from knee cartilages of OA patients were stimulated with IL-1β for 4 hours and RNA was isolated. One microgram of total RNA was polyadenylated and converted to cDNA and miRNA microarray was performed. Seven hundred thirty five oligos were used, corresponding to 470 well-annotated human miRNA sequences and 265 potential miRNAs that were identified recently. mRNA microarray was performed simultaneously using the RNA samples that were used for miRNA array. Both sequence and expression information was used to identify regulatory relationship between miRNA and mRNA pairs. Results. Expression profiling of miRNA extracted from IL-1β treated chondrocytes identified 25 miRNA which showed differential expression. We also identified 7190 mRNAs differentially regulated by IL-1β treatment. Among the 25 miRNAs differentially regulated, 13 miRNAs had targets searched by MiRANDA scheme. By combining target search and miRNA-mRNA pairing, we could identify 1043 miRNA-mRNA target pairs. MiR-200a was found to be expressed in human OA and normal cartilages, with downregulation in OA lesion cartilages. Conclusion. It is suggested that miRNA may play a role in the regulation of cartilage degradation in OA.

Down-regulation of Intestinal Apical Calcium Entry Channel TRPV6 by Ubiquitin E3 Ligase Nedd4-2

Nedd4-2 is an archetypal HECT ubiquitin E3 ligase that disposes target proteins for degradation. Because of the proven roles of Nedd4-2 in degradation of membrane proteins, such as epithelial Na(+) channel, we examined the effect of Nedd4-2 on the apical Ca(2+) channel TRPV6, which is involved in transcellular Ca(2+) transport in the intestine using the Xenopus laevis oocyte system. We demonstrated that a significant amount of Nedd4-2 protein was distributed to the absorptive epithelial cells in ileum, cecum, and colon along with TRPV6. When co-expressed in oocytes, Nedd4-2 and, to a lesser extent, Nedd4 down-regulated the protein abundance and Ca(2+) influx of TRPV6 and TRPV5, respectively. TRPV6 ubiquitination was increased, and its stability was decreased by Nedd4-2. The Nedd4-2 inhibitory effects on TRPV6 were partially blocked by proteasome inhibitor MG132 but not by the lysosome inhibitor chloroquine. The rate of TRPV6 internalization was not significantly altered by Nedd4-2. The HECT domain was essential to the inhibitory effect of Nedd4-2 on TRPV6 and to their association. The WW1 and WW2 domains interacted with TRPV6 terminal regions, and a disruption of the interactions by D204H and D376H mutations in the WW1 and WW2 domains increased TRPV6 ubiquitination and degradation. Thus, WW1 and WW2 may serve as a molecular switch to limit the ubiquitination of TRPV6 by the HECT domain. In conclusion, Nedd4-2 may regulate TRPV6 protein abundance in intestinal epithelia by controlling TRPV6 ubiquitination.

Angiopoietin-like Protein 3 Inhibits Lipoprotein Lipase Activity through Enhancing Its Cleavage by Proprotein Convertases

Lipoprotein lipase (LPL)-mediated lipolysis of triglycerides is the first and rate-limiting step in chylomicron/very low density lipoprotein clearance at the luminal surface of the capillaries. Angiopoietin-like protein 3 (ANGPTL3) is shown to inhibit LPL activity and plays important roles in modulating lipoprotein metabolism in vivo. However, the mechanism by which it inhibits LPL activity remains poorly understood. Using cell-based analysis of the interaction between ANGPTL3, furin, proprotein convertase subtilisin/kexin type 5 (PCSK5), paired amino acid converting enzyme-4 (PACE4), and LPL, we demonstrated that the cleavage of LPL by proprotein convertases is an inactivation process, similar to that seen for endothelial lipase cleavage. At physiological concentrations and in the presence of cells, ANGPTL3 is a potent inhibitor of LPL. This action is due to the fact that ANGPTL3 can enhance LPL cleavage by endogenous furin and PACE4 but not by PCSK5. This effect is specific to LPL but not endothelial lipase. Both N- and C-terminal domains of LPL are required for ANGPTL3-enhanced cleavage, and the N-terminal domain of ANGPTL3 is sufficient to exert its effect on LPL cleavage. Moreover, ANGPTL3 enhances LPL cleavage in the presence of either heparan sulfate proteoglycans or glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1). By enhancing LPL cleavage, ANGPTL3 dissociates LPL from the cell surface, inhibiting both the catalytic and noncatalytic functions of LPL. Taken together, our data provide a molecular connection between ANGPTL3, LPL, and proprotein convertases, which may represent a rapid signal communication among different metabolically active tissues to maintain energy homeostasis. These novel findings provide a new paradigm of specific protease-substrate interaction and further improve our knowledge of LPL biology.