Increase In Relative Expression Research Articles

Plasma miR-21 and miR-31 as Predictive Biomarkers for Evaluation of Therapeutic Efficacy in Metastatic Colorectal Cancer

Summary Introduction. Colorectal cancer (CRC) is the third most common cancer worldwide. Evaluation of patient response to the applied therapy regime is still challenging. Routine laboratory tests during follow-up do not provide necessary information on therapy efficacy. To improve life expectancy and quality of life for patient with CRC ongoing researches is concentrated to discovering new, reliable biomarkers which could contribute to define patients prognosis and choice of therapy. One of topical research field for searching new biomarkers is mi-RNS. Aim of the study. The aim of this study was to analyze relative expression changes of miR-21 and miR-31 as potential biomarkers for evaluation of therapy efficacy in mCRC. Material and methods. In the present study seven patients with mCRC diagnosis were included. After surgery all patients received first line therapy with oxaliplatin. Blood samples for present study were collected every 2-3 weeks. Relative expression of miR-31 and miR-21 assessed with real time PCR. Data analysis carried out by delta delta Ct (ΔΔCt) algorithm. Correlation analysis was performed by R programme ver. 3.1.2 using Pearson correlation coefficient. Results. Our results in the correlation analysis for CEA serum level and relative expression level of miR-21 and miR-31 did not confirm significant correlation. MiR-31 showed increased expression soon after administration of therapy with a drop of relative expression closer to the CDP I. Acquired results revealed trend of increase in relative expression of miR-21 in PFS II compared to PFS I. Correlation analysis for miR-31 and miR-21 did not reach statistical reliability. Conclusions. The relative expression alteration pattern of miR-31 and miR-21 in plasma during the therapy is promising biomarker for evaluation of therapy efficacy. These findings need to be validated in large cohort sample set.

Effects of hypoxia on anabolic and catabolic gene expression and DNA methylation in OA chondrocytes.

BackgroundCartilage is an avascular and aneural tissue. Chondrocytes thrive in this restricted environment of low oxygen tension and poor nutrient availability which has led to suggestions that hypoxia may be a protective mechanism against the development of osteoarthritis (OA). There is also a growing body of evidence to support the role of epigenetic factors in the pathogenesis of OA. However, few studies have investigated the epigenetic-OA process within a hypoxic environment. The current study has investigated the effects of hypoxia on gene expression and DNA methylation of anabolic and catabolic genes involved in the pathogenesis of OA.MethodsChondrocytes extracted from OA femoral heads were incubated in normoxia and hypoxia (20% and 2% oxygen concentrations respectively). Interleukin 1-beta (IL-1β) plus oncostatin M (OSM), 5-azadeoxycytidine (5-aza-dC) or media alone (control) were added twice weekly to the incubated samples. After 5 weeks, levels of Collagen type IX (COL9A1), IL1B, and matrix metalloproteinase-13 (MMP13) gene expression were measured using SYBR Green-based qRT-PCR and were correlated with methylation status analysed by pyrosequencing methodology.ResultsHypoxia resulted in a >50-fold and >10-fold increase in relative expression of COL9A1 and IL1B respectively. This was inversely correlated to the DNA methylation status of these genes. Expression of MMP13 was reduced at 2% oxygen tension in control cells. Relative expression of MMP13 increased in cells stimulated with IL-1β and 5-aza-dC in normoxic conditions, and this effect was eliminated at low oxygen tension although no correlation with methylation status was observed.ConclusionsThese findings demonstrate a role for hypoxia in the regulation of anabolic and catabolic gene expression and the influence of changes in DNA methylation. These results further support the role of epigenetics in OA and, critically, highlight the complex relationship between the physiological environment of cartilaginous cells and the osteoarthritic process with implications for therapeutic intervention and our understanding of OA pathophysiology.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-431) contains supplementary material, which is available to authorized users.

Influence of ferric iron on gene expression and rhamnolipid synthesis during batch cultivation of Pseudomonas aeruginosa PAO1.

Bioprocesses based on sustainable resources and rhamnolipids in particular have become increasingly attractive in recent years. These surface-active glycolipids with various chemical and biological properties have diverse biotechnological applications and are naturally produced by Pseudomonas aeruginosa. Their production, however, is tightly governed by a complex growth-dependent regulatory network, one of the major obstacles in the way to upscale production. P. aeruginosa PAO1 was grown in shake flask cultures using varying concentrations of ferric iron. Gene expression was assessed using quantitative PCR. A strong increase in relative expression of the genes for rhamnolipid synthesis, rhlA and rhlC, as well as the genes of the pqs quorum sensing regulon was observed under iron-limiting conditions. Iron repletion on the other hand caused a down-regulation of those genes. Furthermore, gene expression of different iron regulation-related factors, i.e. pvdS, fur and bqsS, was increased in response to iron limitation. Ensuing from these results, a batch cultivation using production medium without any addition of iron was conducted. Both biomass formation and specific growth rates were not impaired compared to normal cultivation conditions. Expression of rhlA, rhlC and pvdS, as well as the gene for the 3-oxo-C12-HSL synthetase, lasI, increased until late stationary growth phase. After this time point, their expression steadily decreased. Expression of the C4-HSL synthetase gene, rhlI, on the other hand, was found to be highly increased during the entire process.

Molecular cloning and characterization of cDNA encoding a Translocon-Associated Protein (TRAPδ) from the root-lesion nematode Pratylenchus goodeyi

The translocon-associated protein (TRAP) complex comprises four subunits (α, β, γ, δ) and is located in the endoplasmic reticulum membrane at translocation sites. The TRAP complex is required for the efficient translocation of substrates and to correct or eliminate misfolded proteins. In this study, we described the cloning and characterization of a cDNA encoding a TRAP from the phytoparasitic nematode Pratylenchus goodeyi (Pg). The full-length cDNA had an estimated size of 690 bp and encodes a 177 amino acid peptide. The deduced protein after sequence analysis codes for TRAPδ subunit homologous to TRAPδ from other nematodes. The Pg-TRAPδ had a signal peptide indicating a possible involvement in the transport and binding of other proteins at the endoplasmic reticulum membrane. The increase in relative expression of Pg-trapδ, assessed by semi-quantitative PCR, was induced over time in nematodes exposed to a nematostatic/nematicide extract of Solanum nigrum, suggesting that this gene product might be influenced by response mechanisms to stress in P. goodeyi. This is the first report of the cloning and characterization of trap cDNA from plant endoparasitic nematodes.

Development of interspecies testicular germ-cell transplantation in flatfish

Interspecific testicular germ cell (TGC) transplantation was investigated in two commercial flatfish species. Testes from donor species (Senegalese sole) were evaluated using classical histological techniques (haematoxylin-eosin staining and haematoxylin-light green-orange G-acid fuchsine staining), in situ hybridisation and immunohistochemical analysis. Both Ssvasa1-2 mRNAs and SsVasa protein allowed the characterisation of TGCs, confirming the usefulness of the vasa gene in the detection of Senegalese sole TGCs. Xenogenic transplants were carried out using TGCs from one-year-old Senegalese sole into turbot larvae. Propidium iodide-SYBR-14 and 4',6'-diamidino-2-phenylindole (DAPI) staining showed that 87.98% of the extracted testicular cells were viable for microinjection and that 15.63% of the total recovered cells were spermatogonia. The vasa gene was characterised in turbot recipients using cDNA cloning. Smvasa mRNA was confirmed as a germ cell-specific molecular marker in this species. Smvasa expression analysis during turbot ontogeny was carried out before Senegalese sole TGC transplants into turbot larvae. Turbot larvae at 18 days after hatching (DAH) proved to be susceptible to manipulation procedures. High survival rates (83.75±15.90-100%) were obtained for turbot larvae at 27, 34 and 42 DAH. These data highlight the huge potential of this species for transplantation studies. Quantitative PCR was employed to detect Senegalese sole vasa mRNAs (Ssvasa1-2) in the recipient turbot larvae. The Ssvasa mRNAs showed a significant increase in relative expression in 42-DAH microinjected larvae three weeks after treatment, showing the proliferation of Senegalese sole spermatogonia in transplanted turbot larvae.

First molecular evidence of cross-species induction of metalloprotease gene expression in Vibrio strains pathogenic for Pacific oyster Crassostrea gigas involving a quorum sensing system

The aim of that study was to explore the hypothesis that a quorum sensing mechanism modulates metalloprotease gene expression of two Vibrio species pathogenic for oyster, at intraspecific (Vibrio splendidus) and interspecific (V. splendidus affecting V. aestuarianus) levels. Metalloprotease activities (Vsm for V. splendidus LGP32 and Vam for V. aestuarianus 02/041) and growth curves obtained by real-time PCR assay revealed cell density-dependent metalloprotease inductions triggered between 12h and 16h of culture. A quorum sensing assay was then developed using a conditioned medium prepared from supernatant of a mutant strain of V. splendidus LGP32 unable to produce metalloprotease. Specific real-time qPCR assays targeting metalloprotease genes vsm and vam were performed. A relative increase in expression was observed for vsm and, particularly, vam in the presence of the conditioned medium, probably controlled by a quorum sensing system. The study revealed intraspecific and interspecific modulation of expression in metalloprotease genes vsm and vam in V. splendidus and V. aestuarianus.Our experiments showed for the first time that V. splendidus is able to produce an autoinducer-like substance that displays intra- and interspecific effects on the expression of two different metalloprotease genes: vsm and vam.

The conserved surface M-protein SiMA of Streptococcus iniae is not effective as a cross-protective vaccine against differing capsular serotypes in farmed fish

Streptococcus iniae causes invasive infections in fresh and saltwater fish and occasional zoonoses. Vaccination against S. iniae is complicated by serotypic variation determined by capsular polysaccharide. A potential target for serologically cross-protective vaccines is the M-like protein SiMA, an essential virulence factor in S. iniae that is highly conserved amongst virulent strains. The present study determined how SiMA is regulated and investigated potential as a cross-protective vaccine for fish. Electrophoretic mobility shift suggested that SiMA is regulated by the multigene regulator Mgx via a binding site in the −35 region of the simA promoter. Moreover, expression of simA and mgx was highly correlated, with the highest level of simA and mgx expression during exponential growth under iron limitation (20-fold increase in relative expression compared to growth in Todd-Hewitt broth). Based on these results, a vaccination and challenge experiment was conducted in barramundi (Lates calcarifer) to determine whether SiMA is protective against S. iniae infection and cross-protective against a different capsular serotype. The challenge resulted in 60% mortality in control fish. Formalin-killed bacterins prepared from the challenge strain resulted in 100% protection, whereas bacterins prepared from a serotypically heterologous strain resulted in significantly reduced protection, even when culture conditions were manipulated to optimise SiMA expression. Moreover, recombinant SiMA protein was not protective against the challenge strain in spite of eliciting specific antibody response in vaccinated fish. Specific antibody did not increase oxidative activity or phagocytosis by barramundi macrophages. Indeed incubating S. iniae with antisera significantly reduced phagocytosis. Lack of specific-antibody mediated opsonisation in spite of 100% protection against challenge with the homologous vaccine suggests that other immune parameters result in protection of challenged fish.

Mechanisms for Photoinactivation of Enterococcus faecalis in Seawater

Field studies in fresh and marine waters consistently show diel fluctuations in concentrations of enterococci, indicators of water quality. We investigated sunlight inactivation of Enterococcus faecalis to gain insight into photoinactivation mechanisms and cellular responses to photostress. E. faecalis bacteria were exposed to natural sunlight in clear, filtered seawater under both oxic and anoxic conditions to test the relative importance of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. Multiple methods were used to assess changes in bacterial concentration, including cultivation, quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, LIVE/DEAD staining using propidium iodide (PI), and cellular activity, including ATP concentrations and expression of the superoxide dismutase-encoding gene, sodA. Photoinactivation, based on numbers of cultivable cells, was faster in oxic than in anoxic microcosms exposed to sunlight, suggesting that oxygen-mediated photoinactivation dominated. There was little change in qPCR signal over the course of the experiment, demonstrating that the nucleic acid targets were not damaged to a significant extent. The PMA-qPCR signal was also fairly stable, consistent with the observation that the fraction of PI-permeable cells was constant. Thus, damage to the membrane was minimal. Microbial ATP concentrations decreased in all microcosms, particularly the sunlit oxic microcosms. The increase in relative expression of the sodA gene in the sunlit oxic microcosms suggests that cells were actively responding to oxidative stress. Dark repair was not observed. This research furthers our understanding of photoinactivation mechanisms and the conditions under which diel fluctuations in enterococci can be expected in natural and engineered systems.

Abstract PR16: Important bidirectional function in the Notch-Delta signaling in pancreatic carcinogenesis and metastasis

Abstract Pancreatic cancer is a dismal disease with a median survival of less than 6 months. Its high mortality is due to aggressive development and early lymphatic and hematogenic dissemination. Notch signaling has been shown to regulate pancreatic carcinogenesis; however, a specific role of the Notch ligands has not been extensively studied thus far. Here we investigate the role of Delta1 ligand in pancreatic carcinogenesis and metastasis. Our study using a reporter mouse indicates that Delta1 is mainly expressed in centroacinar and ductal cells as well as in pancreatic intraepithelial neoplasia (PanIN) lesions. Expression in those vulnerable cell compartments and preneoplastic lesions imply relevance for carcinogenesis. Moreover, during cancer development, Delta1 shows the highest relative increase in expression among all Notch ligands suggesting it predominantly functions in Notch pathway activation. Its potentially important role in tumorigenesis was investigated using a KrasG12D-driven pancreatic carcinogenesis mouse model inbreed with a Delta1LacZ/+ haploinsufficient mouse. In KrasG12D;Delta1LacZ/+ mice we observed a delay in PanIN progression and increased development of mucinous cystic neoplasia. Obtained results support our hypothesis. Moreover, in vitro data indicates that Delta1 is critical for Notch2 activation that directly regulates pancreatic cancer progression via cMyc induction. We also analyzed expression and role of the intracellular domain of Delta1 (Delta1-IC). We found that Delta1-IC is accumulated in pancreatic cancer distal metastasis. Our investigations strongly suggest that Delta1-IC can enhance metastatic potential by interaction with transducers of TGFβ signaling: Smad3 and Smad4. We found that Delta1-IC by co-binding to Smad3-regulated promoter proteins can increase TGFβ-induced transcriptional changes in the cell leading to increased cellular motility and EMT capacity. We establish that nuclear accumulation of Delta1-IC correlates with patients' poor outcome and presents of metastasis as well as tumor stage. Our investigation proofs the importance of Notch signaling and points to its bidirectional function in pancreas carcinogenesis and metastasis. This abstract is also presented as Poster A62. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR16.

Smooth muscle phenotypic diversity is mediated through alterations in Myocardin gene splicing

Myocardin (MYOCD) is a smooth and cardiac muscle-specific transcriptional coactivator that is required for the proper expression of contraction-related genes. Through its function to transactivate effector genes, MYOCD plays an essential role in mediating the switch between contractile and non-contractile phenotypes, particularly in smooth muscle cells (SMC). There are at least two known transcript variants of MYOCD that are expressed in SMC, differing only by the presence (+) or absence (Δ) of Exon 11. To date, no functional role has been assigned to the domain encoded by Exon 11, nor have any notable differences between the ability of each isoform to activate contraction-related genes been observed. In this study we compared sequences for Exon 11 among several mammalian species and identified a highly conserved, putative target sequence for glycogen synthase kinase 3 (GSK3) phosphorylation, suggesting a regulatory role for Exon 11 that can be modulated by alternative splicing. The function of Exon 11 was investigated by altering MYOCD splice selection in cultured porcine SMC with small interfering RNAs (siRNA) and specific chemical inhibitors, resulting in a relative increase in expression of ΔExon 11 variants in the endogenous pool of MYOCD mRNA. The relative increase in ΔExon 11 mRNAs correlated with a reduction of contractile phenotype in the porcine SMC as evidenced by morphological assessment and molecular analysis of effector genes. Together, these data suggest that MYOCD ΔExon 11 may participate in modulating SMC phenotype, potentially acting as a dominant-negative repressor of contraction-related genes.

Low Levels of Physical Activity Increase Metabolic Responsiveness to Cold in a Rat (Rattus fuscipes)

BackgroundPhysical activity modulates expression of metabolic genes and may therefore be a prerequisite for metabolic responses to environmental stimuli. However, the extent to which exercise interacts with environmental conditions to modulate metabolism is unresolved. Hence, we tested the hypothesis that even low levels of physical activity are beneficial by improving metabolic responsiveness to temperatures below the thermal neutral zone, thereby increasing the capacity for substrate oxidation and energy expenditure.Methodology/Principal FindingsWe used wild rats (Rattus fuscipes) to avoid potential effects of breeding on physiological phenotypes. Exercise acclimation (for 30 min/day on 5 days/week for 30 days at 60% of maximal performance) at 22°C increased mRNA concentrations of PGC1α, PPARδ, and NRF-1 in skeletal muscle and brown adipose tissue compared to sedentary animals. Lowering ambient temperature to 12°C caused further increases in relative expression of NRF-1 in skeletal muscle, and of PPARδ of brown adipose tissue. Surprisingly, relative expression of UCP1 increased only when both exercise and cold stimuli were present. Importantly, in sedentary animals cold acclimation (12°C) alone did not change any of the above variables. Similarly, cold alone did not increase maximum capacity for substrate oxidation in mitochondria (cytochrome c oxidase and citrate synthase activities) of either muscle or brown adipose tissue. Animals that exercised regularly had higher exercise induced metabolic rates in colder environments than sedentary rats, and temperature induced metabolic scope was greater in exercised rats.Conclusions/SignificancePhysical activity is a necessary prerequisite for the expression of transcriptional regulators that influence a broad range of physiological functions from energy metabolism to cardiovascular function and nutrient uptake. A sedentary lifestyle leads to decreased daily energy expenditure because of a lack of direct use of energy and a muted metabolic response to ambient temperature, which can be reversed even by low levels of physical activity.

Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus.

BackgroundThe effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA) expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea.ResultsThe sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study.ConclusionsOur findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.

The R2 mobile element of Rhynchosciara americana: Molecular, cytological and dynamic aspects

Ribosomal RNA genes are encoded by large units clustered (18S, 5S, and 28S) in the nucleolar organizer region in several organisms. Sometimes additional insertions are present in the coding region for the 28S rDNA. These insertions are specific non-long terminal repeat retrotransposons that have very restricted integration targets within the genome. The retrotransposon present in the genome of Rhynchosciara americana, RaR2, was isolated by the screening of a genomic library. Sequence analysis showed the presence of conserved regions, such as a reverse transcriptase domain and a zinc finger motif in the amino terminal region. The insertion site was highly conserved in R. americana and a phylogenetic analysis showed that this element belongs to the R2 clade. The chromosomal localization confirmed that the RaR2 mobile element was inserted into a specific site in the rDNA gene. The expression level of RaR2 in salivary glands during larval development was determined by quantitative RT-PCR, and the increase of relative expression in the 3P of the fourth instar larval could be related to intense gene activity characteristic of this stage. 5'-Truncated elements were identified in different DNA samples. Additionally, in three other Rhynchosciara species, the R2 element was present as a full-length element.

ZD4054: a specific endothelin A receptor antagonist with promising activity in metastatic castration-resistant prostate cancer

Background: Overexpression of the endothelin A (ETA) receptor has been found in a number of human cancer cell lines. Activation of the ETA receptor by endothelin-1 (ET-1) promotes cell proliferation and survival in these tumors, whereas activation of the endothelin B (ETB) receptor results in an opposing effect. Therefore, blockade of ETA may have antitumor effects, while sparing ETB-mediated effects such as induction of apoptosis and clearance of ET-1. Objective: ZD4054 is an orally bioavailable, specific ETA antagonist currently being investigated in prostate cancer. In receptor-binding studies, ZD4054 only bound to ETA, with no binding detected towards ETB. Prostate cancer cell lines are known to produce ET-1 and there is a relative increase in expression of ETA to ETB in these cancers. There is also an association of greater ETA expression in higher grade versus lower grade tumors, suggesting that ETA may be involved in the malignant transformation process. As ET-1 may also mediate nociceptive effects and osteoblastic effects, there is much interest in clinically assessing ZD4054 in prostate cancer. Methods: The data describing the endothelin axis, the role of ETA and ETB in malignancy, and the effects of ETA antagonist ZD4054 in prostate cancer, as demonstrated in preclinical and clinical studies, are reviewed. Results: Further investigation of ZD4054 in prostate cancer is warranted, and Phase III trials are already planned in patients with non-metastatic castrate-resistant prostate cancer (CRPC) with rising prostate specific antigen values, metastatic (asymptomatic) CRPC, and in metastatic CRPC in combination with docetaxel, assessing either differences in progression-free survival and overall survival or overall survival alone.

Eotaxin-2 and Colorectal Cancer: A Potential Target for Immune Therapy

To study the production of chemokines by colorectal hepatic metastases. Biopsies of resected colorectal hepatic metastases and nonneoplastic adjacent liver tissue were screened for chemokines using protein arrays and results were confirmed by ELISA and immunohistochemistry. Two chemokines, eotaxin-2 and MCP-1, were found at elevated levels within the tumor biopsy compared with adjacent liver. The relative increase in expression from tumor was much higher for eotaxin-2 than MCP-1, with 10 of 25 donors having a >100-fold increase in expression compared with 0 of 24 donors for MCP-1. In a parallel analysis, eotaxin-2 was also found at elevated levels in the tumor region of primary colorectal cancer biopsies. Immunohistochemical staining indicated that carcinoembryonic antigen-positive tumor cells stained strongly for eotaxin-2, implicating these cells as the predominant source of the chemokine. In vitro studies confirmed that several colorectal tumor lines produce eotaxin-2 and that secretion of this chemokine could be depressed by IFN-gamma and enhanced by the Th2-type cytokines interleukin-4 and interleukin-13. Jurkat T cells were engineered to express the receptor for eotaxin-2 (CCR3). These cells effectively migrated in response to eotaxin-2 protein, suggesting that immune cells gene modified to express a chemokine receptor may have improved abilities to home to tumor. Taken together, these observations confirm eotaxin-2 as a chemokine strongly associated with primary and metastatic tumors of colorectal origin. Furthermore, the importance of this result may be a useful tool in the development of targeted therapeutic approaches to colorectal tumors.

Prenatal high-salt diet in the Sprague-Dawley rat programs blood pressure and heart rate hyperresponsiveness to stress in adult female offspring

Several animal models have been developed to study fetal programming of hypertension. One model involves feeding high-salt (HS) diet to rats before and during pregnancy, during lactation, and after weaning for 10 days. In the present investigation, we limited HS diet to the prenatal period in an attempt to find a narrower critical window for fetal programming. The HS diet did not result in low-birth weight offspring. In the adult offspring, radiotelemetry was used to assess blood pressure and heart rate in the conscious unstressed state. As adults, the HS offspring were not hypertensive compared with normal-salt (NS) control animals. However, the pressor and tachycardic responses to 1-h of restraint were significantly enhanced in HS female offspring, and recovery after restraint was delayed. This was accompanied by an increase in relative expression of corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus during basal and stressed conditions. There was no augmented stress response or relative increase in CRH mRNA in adult HS male offspring. When challenged with 1 wk of 8% NaCl diet as adults, neither HS male nor female offspring exhibited salt sensitivity compared with NS groups. These data show that a high-salt diet limited to the prenatal period is not sufficient to program hypertension in adult offspring. However, this narrower critical period is sufficient to imprint a lasting hyperresponsiveness to stress, at least in adult female offspring. These data indicate that excessive maternal salt intake during pregnancy can adversely affect the cardiovascular health of adult offspring.

177 EXPRESSION PROFILING OF HISTONE-MODIFYING GENES IN BOVINE PREIMPLANTATION EMBRYOS

Global activation of the embryonic genome marks one of the most important aspects of early embryonic development and coincides with both global DNA and histone modifications necessary to control gene expression for further development. In bovine preimplantation embryos, epigenetic reprogramming occurs between fertilization and the blastocyst stage. De novo DNA methylation from the 8-cell stage to the morula represents activation of DNA methyltransferases and histone-modifying genes; however, it is still unclear how these critical events of early development and differentiation are regulated. In this preliminary study, we used quantitative real-time PCR (qRT-PCR) to examine stage-specific expression in bovine gametes (testis and ova) and preimplantation embryos of eight histone-modifying genes: LSH1 (which associates with both DNA and histone methyltransferases), LSD1 (a histone demethylase), SUV39H1, SUV420H1, SETB1, SUZ12, SMYD3, and G9a (all representative histone methyltransferases). Bovine ova and embryos were produced via in vitro maturation, fertilization, and culture from a single pool of ova as per standard laboratory protocol. Testes were harvested from a mature bull and immediately snap frozen in liquid nitrogen. RNA was isolated from testis, and groups of 10–15 in vitro-matured bovine ova or in vitro-produced embryos at the 2-, 8-cell, morula, and blastocyst stages and stored at –80�C until further use. cDNA was generated from each RNA sample using Superscript II" reverse transcriptase (Invitrogen, Carlsbad, CA, USA) under identical PCR conditions. Relative gene expression from each RNA sample was calculated in triplicate using the ΔΔCt method with both SYBR� Green and Taqman� qRT-PCR methods (Applied Biosystems, Foster City, CA, USA) and normalized to endogenous bovine GAPDH expression as per standard protocol. Due to excessive variation in a few of the qRT-PCR reactions, consistent data were collected for only SETB1, SUV39H1, SUV420H1, and G9a. SETB1 and G9a shared a similar expression pattern, with both exhibiting a dramatic relative increase in transcript level near the 8-cell stage and reduction to basal levels thereafter. Also, SUV39H1 and SUV420H1 were similar, showing a gradual increase in relative expression from the oocyte to the 8-cell stage, followed by a drop to low levels by the morula stage. These data indicate that several histone-modifying genes are expressed in distinct patterns during the critical period of zygotic genome activation and establishment of the epigenome in early preimplantation bovine embryos. Continuing studies will determine specific gene function through shRNA-induced silencing of histone-modifying genes and their role in control of gene expression.

Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion

Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the stationary phase during electron donor depletion. In addition to temporal transcriptomics, total protein, carbohydrate, lactate, acetate, and sulfate levels were measured. The microarray data were examined for statistically significant expression changes, hierarchical cluster analysis, and promoter element prediction and were validated by quantitative PCR. As the cells transitioned from the exponential phase to the stationary phase, a majority of the down-expressed genes were involved in translation and transcription, and this trend continued at the remaining times. There were general increases in relative expression for intracellular trafficking and secretion, ion transport, and coenzyme metabolism as the cells entered the stationary phase. As expected, the DNA replication machinery was down-expressed, and the expression of genes involved in DNA repair increased during the stationary phase. Genes involved in amino acid acquisition, carbohydrate metabolism, energy production, and cell envelope biogenesis did not exhibit uniform transcriptional responses. Interestingly, most phage-related genes were up-expressed at the onset of the stationary phase. This result suggested that nutrient depletion may affect community dynamics and DNA transfer mechanisms of sulfate-reducing bacteria via the phage cycle. The putative feoAB system (in addition to other presumptive iron metabolism genes) was significantly up-expressed, and this suggested the possible importance of Fe2+ acquisition under metal-reducing conditions. The expression of a large subset of carbohydrate-related genes was altered, and the total cellular carbohydrate levels declined during the growth phase transition. Interestingly, the D. vulgaris genome does not contain a putative rpoS gene, a common attribute of the delta-Proteobacteria genomes sequenced to date, and the transcription profiles of other putative rpo genes were not significantly altered. Our results indicated that in addition to expected changes (e.g., energy conversion, protein turnover, translation, transcription, and DNA replication and repair), genes related to phage, stress response, carbohydrate flux, the outer envelope, and iron homeostasis played important roles as D. vulgaris cells experienced electron donor depletion.

267 CLASSICAL AND NON-CLASSICAL MAJOR HISTOCOMPATABILITY COMPLEX CLASS I GENE EXPRESSION IN IN VITRO-DERIVED BOVINE EMBRYOS

The major histocompatibility complex class I (MHC-I) genes have been shown to play a role in the establishment of pregnancy in humans and mice. In particular, the secretion of the soluble isoform of the human non-classical HLA-G gene during in vitro embryo culture has been linked to higher pregnancy rates following embryo transfer. In contrast to the situation in humans, knowledge of the bovine MHC-I is quite limited. The objective of the current study was to characterize MHC-I gene expression during bovine pre-implantation development in vitro. Immature bovine oocytes were matured, fertilized, and cultured in vitro. Three replicate pools of 20 samples were collected at immature and mature oocyte, presumptive zygote, 2–4 cell, 8–16 cell, morula, blastocyst, and hatched blastocyst stages. Relative MHC-I mRNA expression levels were quantified using quantitative real-time PCR across pre-implantation development with a generic primer pair for global classical and non-classical MHC-I gene expression and primer pairs for two known non-classical MHC-I genes, N*50001 and N*50101 (NCBI accession numbers: X80936 and AY188807), cDNA sequencing, and wholemount immunocytochemistry with a Pan MHC-I-protein antibody (ILA88). MHC-I mRNA transcripts amplified by the generic primers were detected in greatest abundance in the oocyte and cumulus cells of immature oocytes (P < 0.05); following maturation, the expression levels decreased by 5-fold but remained stable until the 2- to 4-cell stage. Levels remained low until the morula and blastocyst stages when a small increase in relative expression was detected. Sequence analysis of oocyte and blastocyst PCR products amplified by the generic primers was carried out. The generic primers amplified a total of 6 discrete sequences, which are listed on the bovine MHC database (http://www.ebi.ac.uk/ipd/mhc/bola): (i) N*02601, (ii) N*02301, (iii) N*01301, (iv) N*04001, (v) N*01601, and (vi) non-classical N*5003. The mRNA expression pattern of Gene X differed from the global MHC-I mRNA profile in that mRNA abundance was highest in the matured oocytes (P < 0.05), after which abundance decreased 5-fold in the zygote and was not detected in the subsequent embryo stages until the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were Gene X. Relative HD15 mRNA expression was highest in immature oocytes (P < 0.05); however, there was a steep decrease in abundance following oocyte maturation (P < 0.05), followed by a transient increase at the presumptive zygote stage (P < 0.05). Thereafter, expression levels remained low up to the morula and blastocyst stages. Sequence analysis confirmed that the PCR products were HD15. MHC-I protein expression was detected in immature and mature oocytes and their surrounding granulosa cells, in 2- and 4-cell embryos, and in blastocysts. In conclusion, these data demonstrate that classical and non-classical MHC-I genes are expressed in bovine oocytes and embryos at both the mRNA and the protein levels, and the expression pattern is gene- and stage-specific.

Treatment With Simvastatin Suppresses the Development of Experimental Abdominal Aortic Aneurysms in Normal and Hypercholesterolemic Mice

To determine if treatment with hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) can influence the development of experimental abdominal aortic aneurysms (AAAs). AAAs are associated with atherosclerosis, chronic inflammation, and matrix metalloproteinase (MMP)-mediated connective tissue destruction. Because statins exert antiinflammatory activities independent of their lipid-lowering effects, these agents may help suppress aneurysmal degeneration. C57Bl/6 wild-type and hypercholesterolemic apoE-deficient mice underwent transient perfusion of the aorta with elastase followed by subcutaneous treatment with either 2 mg/kg simvastatin per day or vehicle. Aortic diameter (AD) was measured before and 14 days after elastase perfusion. The extent of aortic dilatation (DeltaAD) was determined with AAAs defined as DeltaAD >100%. Wild-type mice treated with simvastatin exhibited a 21% reduction in DeltaAD and a 33% reduction in AAAs compared with vehicle-treated controls. Suppression of AAAs in simvastatin-treated mice was associated with preservation of medial elastin and vascular smooth muscle cells, as well as a relative reduction in aortic wall expression of MMP-9 and a relative increase in expression of TIMP-1. In hypercholesterolemic apoE-deficient mice, treatment with simvastatin was associated with a 26% reduction in DeltaAD and a 30% reduction in AAAs. Treatment with simvastatin had no effect on serum cholesterol levels in either normal or hypercholesterolemic mice. Treatment with simvastatin suppresses the development of experimental AAAs in both normal and hypercholesterolemic mice. The mechanisms of this effect are independent of lipid-lowering and include preservation of medial elastin and smooth muscle cells, as well as altered aortic wall expression of MMPs and their inhibitors.