Abundance Of Specific mRNAs Research Articles

T helper cells exhibit a dynamic and reversible 3'-UTR landscape.

3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

MRNA decapping activators Pat1 and Dhh1 regulate transcript abundance and translation to tune cellular responses to nutrient availability.

We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors. Although the Environmental Stress Response (ESR) is activated in dhh1Δ and pat1Δ mutants, hundreds of non-ESR transcripts are elevated in a manner indicating cumulative repression by Pat1 and Dhh1 in wild-type cells. These mRNAs show bothreduced decapping and diminished transcription in the mutants, indicating that impaired mRNA turnover drives transcript derepression in cells lacking Dhh1 or Pat1. mRNA degradation stimulated by Dhh1/Pat1 is not dictated by poor translation nor enrichment for suboptimal codons. Pat1 and Dhh1 also collaborate to reduce translation and protein production from many mRNAs. Transcripts showing concerted translational repression by Pat1/Dhh1 include mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress numerous transcripts involved in respiration, catabolism of non-preferred carbon or nitrogen sources, or autophagy; and we obtained evidence for elevated respiration and autophagy in the mutants. Thus, Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation.

Evaluation of the molecular response of corpora lutea to manganese-amino acid complex supplementation in gilts.

Porcine pregnancy establishment and maintenance are dependent on the formation of functional corpora lutea (CL). Manganese (Mn) is critical for CL function as it is a cofactor for Mn superoxide dismutase and enzymes involved in cholesterol synthesis. Previously, we have shown that luteal Mn content increased and luteal progesterone (P4) concentration decreased in the CL of gilts fed diets supplemented with an Mn–amino acid complex (Availa-Mn; Zinpro Corporation) compared with controls fed Mn sulfate. Importantly, serum P4 increased from 0 (estrus onset) to 12 d post estrus (dpe), as expected, but P4 abundance in circulation was not affected by dietary Mn source (P = 0.15). We hypothesized that a more bioavailable Mn source (which results in increased luteal Mn content) would alter the luteal proteome and abundance of mRNA associated with steroid biogenesis during the mid-luteal phase of the estrous cycle. Postpubertal gilts (n = 32) were assigned to one of the four gestation diets. The control diet (CON) contained 20 ppm of supplemental Mn in the form of Mn sulfate. Three additional diets included 20 (TRT1), 40 (TRT2), or 60 (TRT3) ppm of supplemental Mn in the form of a Mn–amino acid complex instead of Mn sulfate. Dietary treatment began at estrus synchronization (approximately 20 d before estrus) and continued through 12 dpe when gilts were euthanized and tissues were collected. Protein and total RNA extracts from the CL were used for proteomic analysis via label-free liquid chromatography with tandem mass spectrometry to assess global protein abundance and quantitative real-time polymerase chain reaction (qRT-PCR) to assess specific mRNA abundance, respectively. Compared with CON, 188, 382, and 401 proteins were differentially abundant (P < 0.10) in TRT1, TRT2, and TRT3, respectively. Gene Ontology enrichment software revealed that proteins involved in P4 signaling and cholesterol synthesis were downregulated in CL of gilts fed Mn–amino acid complex compared with controls. Quantitative RT-PCR showed that relative transcript abundance of genes encoding steroidogenic enzymes (CYP11A1 and StAR) in CL tissue was decreased in gilts from TRT2 compared with CON (P = 0.02), but TRT1 and TRT3 were not affected (P ≥ 0.30). Collectively, these data support our hypothesis that a more bioavailable dietary Mn source may influence luteal function by altering the abundance of protein and mRNA involved in steroidogenesis.

Dietary Na+ Depletion Transcriptionally Up‐regulates BK Channel Mediated K+ Secretion in Proximal Colon

BackgroundElectrogenic K+ secretion and H+‐K+‐ATPase‐mediated electroneutral K+ absorption occur in normal rat proximal and distal colon, respectively. Although both high‐K+ diet and dietary‐Na+ depletion induce K+ secretion in distal colon, only the dietary‐Na+ depletion has been shown to stimulate K+ secretion in proximal colon. Dietary‐Na+ depletion induced both intermediate conductance K+ (IK) and large conductance K+ (BK) channels mediated K+ secretion, while high‐K+ diet induced only the BK channel expression and activity in distal colon. Although K+ secretions are present, the molecular identity of K+ channels that mediate K+ secretion in normal and dietary‐Na+ depleted rat proximal colon is not known.HypothesisIt is hypothesized that dietary‐Na+ depletion may induce K+ secretion by stimulating IK and/or BK channel in proximal colon.AimThis study was initiated to identify the molecular identity of the dietary‐Na+ stimulated K+ secretion in rat proximal colon.MethodsNormal rats (Male Sprague‐Dawly; 200–225g) were fed either normal, high‐K+ or Na+‐depleted diet ad libitum for 6–7 days. Mucosal to serosal (m‐s) and serosal to mucosal (s‐m) 86Rb+ (K+ surrogate) fluxes were measured in stripped proximal colon epithelia mounted under voltage clamp condition. Net K+ fluxes were calculated by subtracting s‐m fluxes from m‐s fluxes. Positive and negative net K+ fluxes represent active absorption and secretion, respectively. IK and BK channel mRNA abundance and protein expression were determined by RT‐qPCR and western blot analyses, respectively. Cell and membrane specific IK and BK channel expressions were determined by immunofluorescence studies.ResultsSimilar to earlier demonstration, active K+ secretion is present in normal proximal colon. Dietary‐Na+ depletion, but not high‐K+ diet stimulated K+ secretion in proximal colon (Normal vs Na+‐depletion: 0.42 ± 0.06 vs 2.33 ± 0.16 μEq/cm2.h; p &lt; 0.001). Mucosal iberiotoxin (IbTX; 10 μM; BK channel blocker) completely inhibited the K+ in normal, while it inhibited only 72% in Na+‐depleted rat proximal colon. Mucosal TRAM34 (10 μM; IK channel blocker) did not affect K+ secretion in normal, but it significantly (22%; p &lt; 0.02) inhibited in Na+‐depleted rat colon. RT‐qPCR analyses revealed increased IK (2.2‐fold) and BK (6.2‐fold) specific mRNA abundance, while western blot analyses confirmed increased expression of IK and BK channel expression. Immunofluorescence studies established that in vitro aldosterone (1 μM) incubation increased both IK and BK protein expression in apical membranes of proximal crypt glands.ConclusionWe conclude that high (~1000 ng/ml; Na+‐depletion), but not low (~300 ng/ml; high‐K+ diet) blood aldosterone level stimulates IK/BK expression to increase K+ secretion in proximal colon.Support or Funding InformationThis study was supported by DK112085 and DK104791 from the National Institute of Health Diabetes and Digestive and Kidney Diseases.

Small molecule supplementation potentiates miRNA mediated direct reprogramming of human fibroblasts into functional hepatocytes

BackgroundHepatic transcription factors such as Hnf4α, Foxa1, Foxa2 or Foxa3 and Gata4 have successfully been used for directly reprogram the fibroblasts into hepatic lineages (iHeps). Although it has several advantages such as devoid of teratoma formation, robust conversion rate and potential in vivo reprograming to repair injured tissues, majority of the direct reprograming that employed genetic manipulations to modify the expression of tissue‐specific transcription factors have limitation in downstream clinical applications. Thus, it is critical to develop a new method for reprograming fibroblasts to hepatocytes.HypothesisWe hypothesized that the combination of small molecules such as epigenetic modifiers and GSK‐inhibitor, and miRNAs could successfully be used in direct differentiation of fibroblasts to iHeps.AimThe aim of this study was to directly reprogram the human fibroblasts into functional iHeps using small molecules that regulate reprogramming of somatic cells and miRNAs.MethodsHepatocyte specific miRNAs were selected from liver fibrosis, regulation of mesenchymal to epithelial transition (MET) and regulation of liver specific transcription factors HNF4α and FOXa2 miRNA datasets. Primary human fibroblasts grown to 80% confluent were transfected with either miRNA cocktail and/or small molecules. Transfected cells were cultured in hepatocyte growth media to generate iHeps. Albumin, E‐cad, HNF4α and FOXa2 expressions were detected by flow cytometry, immunostaining and qPCR analyses to establish iHeps. The iHeps mixed with human vascular endothelial cells (HUVEC) in collagen were implanted in partial‐hepatectomized immune‐compromised mice. Explants collected were characterized 4 weeks following implantation. Human albumin level was estimated by ELISA in iHeps implanted mouse serum.ResultsmiRNA cocktail alone significantly downregulated Snail1 and vimentin, and upregulation E‐cad specific mRNA abundance. Although small molecules do not exert any effect, its supplementation along with miRNA cocktail drastically downregulated Snail1 and vimentin, and upregulated E‐cad abundance. Albumin, E‐cad, HNF4α and FOXa2 expression established iHeps lineage. Integration of iHeps with vascular structures were present in liver (shown by arrows in Figure ), while human albumin (control vs iHeps: 0.78 ± 0.01 vs 63.8 ± 0.2 ng/ml) was detected in serum of mice implanted with iHeps.ConclusionsWe conclude that: 1) the down‐regulation of Snail1 and vimentin, and upregulation E‐cad establish MET; 2) the small molecule supplementation potentiate the miRNA cocktail induced MET; and 3) the direct reprograming of human fibroblast using miRNA along with small molecule supplementation generate a viable and functional iHeps.SpeculationWe speculate that small molecules along with miRNA cocktail could potentially be used to treat liver fibrosis in vivo.Support or Funding InformationFunding was provided by the Institute of Bioengineering and Nanotechnology(Biomedical Research Council, Agency for Science, Technology and Research, Singapore). VMR was supported by NIH grants DK112085 and DK104791.H&amp;E staining of sections of explants harvested from partially hepatectomized mouse modelFigure 1

Plasmodium male gametocyte development and transmission are critically regulated by the two putative deadenylases of the CAF1/CCR4/NOT complex.

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.

Stable Silencing of RUNX1/ETO Induces Expression of a Shortened PU.1 Variant in t(8;21) Kasumi Cells

The t(8;21)(q22;q22) found in about 8% of de novo AML fuses the Runx1 (or AML-1) gene to the ETO (or MTG8) gene. The leukemia-initiating chimeric RUNX1/ETO fusion protein is believed to repress transcription of RUNX1 target genes by ETO-mediated recruitment of transcriptional repressors. Although several studies analyzing short-term depletion of RUNX1/ETO reveal both widespread epigenetic reprogramming and altered protein expression of several signaling molecules depending on RUNX1/ETO (e.g. Ptasinska et al. 2012, Spirin et al. 2014) the molecular mechanisms involved are still not completely understood. We analyzed RUNX1/ETO dependent expression of the myeloid transcription factor PU.1 in t(8;21) Kasumi cells after lentiviral transduction with control or RUNX1/ETO specific shRNAs (shCtrl and shRE). PU.1 is a well known target of RUNX1/ETO being inactivated by the chimeric protein. PU.1 expression was analyzed by immunoblotting of cellular lysates from cells transduced to > 99%. RUNX1/ETO is depleted upon shRE expression, and shRE cells exhibit reduced proliferation, less apoptotic cell death and reduced clonal growth in limiting dilution assays as compared to controls. Around day 7 after transduction expression of the full length ~37 kD PU.1 protein is specifically reduced in shRE cells, and a shorter variant of about 22 kD is increasingly expressed starting at day 4. A 20.7 kD PU.1 variant can be predicted from an alternative transcript missing exon 1 encoding the start ATG for full length PU.1. The alternative transcript encodes an ATG in frame in exon 3 with identical 3´-terminal PU.1 sequences. We cloned several PU.1 cDNAs from Kasumi cells and identified and confirmed the alternative transcript missing exon 1. In vitro transcription/translation of cloned PU.1 transcripts generated a protein with C-terminal PU.1 epitopes of the predicted size. However, using PU.1 isoform-specific quantitative real-time RT-PCR we did not find differential changes in isoform-specific mRNA expression in shRE as compared to shCtrl cells with the full length variant always being most abundant. PU.1 expression can be modulated by an antisense RNA regulated by a shared cis-regulatory element (Ebralize et al. 2008) but its expression was similar in shRE and shCtrl cells as determined by quantitative RT-PCR. Since these data suggest that differential protein expression is not due to differential abundance of specific mRNAs we analyzed expression and phosphorylation status of eIF2-α. eIF2-α is involved in translation regulation during cellular stress response by redirecting ribosomes to alternative open reading frames (ORFs) in a phosphorylation dependent manner (for review Young and Wek 2016). eIF2-α phosphorylation increases at ~ 4 days after transduction in shRE but not in shCtrl cells. Interestingly, protein expression of several proteins such as FOXO3, STAT3, STAT5 and C/EBPα is reduced in shRE as compared to shCtrl cells without significant changes in the respective mRNA levels. Furthermore, expression of the RUNX1/ETO targets PU.1, C/EBPα and BCL2 initially increases before final reduction. These data suggest that RUNX1/ETO modulates mRNA translation in addition to its well-known transcriptional and epigenetic effects. Furthermore, RUNX1/ETO targets identified by loss-of function studies may characterize individual candidates as part of a more general response to translational regulation. DisclosuresNo relevant conflicts of interest to declare.

Methods for RNA profiling of gravi-responding plant tissues.

Plant transcriptional responses to gravity stimulation by reorientation are among the fastest measured in any tissue or species. Upon reorientation, changes in abundance of specific mRNAs can be measured within seconds or minutes, for plastid or nuclear encoded genes, respectively. Identifying fast gravity-induced transcripts has been made possible by the development of high-throughput technology for qualitative and quantitative RNA analysis. RNA profiling has undergone further rapid development due to its enormous potential in basic sciences and medical applications. We describe here the current and most widely used methods to profile the changes in an entire transcriptome by high-throughput sequencing of RNA fractions (RNAseq) and single gene transcript analysis using real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).

An arginine-glycine-rich RNA binding protein impacts the abundance of specific mRNAs in the mitochondria of Trypanosoma brucei.

In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo, inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei. TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

Aldosterone transcriptionally regulates large conductance K+ channel β1 (BKβ1)‐ subunit specific mRNA abundance in rat distal colon (LB845)

Background: Large conductance K+ (BK) channels localized on the apical membranes mediate cAMP and aldosterone (“aldo”) activated K+ secretion in rat distal colon. BK channels composed of α (BKα) and β (BKβ) subunits. Four different BKβ (BKβ1, BKβ2, BKβ3 and BKβ4) isoforms have been shown expressed in tissue specific pattern. Recent studies have shown “aldo” transcriptionally up‐regulates BKα‐subunit. The identity of BKβ isoform and its regulation by “aldo” is not known in rat distal colon. Aim:This study was aimed: 1) to identify the BKβ isoform; and 2) to demonstrate whether BKβ subunit also transcriptionally regulated by “aldo” in rat distal colon. Methods: “Aldo” rats (Sprague‐Dawley, 200 ‐ 220 g) were produced by feeding Na‐freed diet ad libitum for 6 ‐ 7 days. Total RNA was purified from epithelial cells isolated form rat proximal and distal colon. BKβ isoform specific fragments were amplified using isoform specific primers and polymerase chain reaction (PCR). Quantitative RT‐PCR (RT‐qPCR) analyses were performed to quantify the expression level of BKβ.isoform in “aldo” rat distal colon. Results: PCR amplified BKβ1, but not BKβ2, BKβ3 and BKβ4 isoform specific fragments in both proximal and distal colon. RT‐qPCR analyses revealed that similar to BKα‐subunit, BKβ1‐subunit specific mRNA expression also enhanced by 3.5‐fold in “aldo” rat distal colon. The P‐values for BKβ1 subunit was less than 0.05 thus indicating a significant change at a 95% confidence. BKβ1‐subunit specific mRNA level is not altered in “aldo” proximal colon. Conclusions: Rat colonic BK channel consists of BKβ1 isoform; and 2) “Aldo” transcriptionally up regulates both BKα‐ and BKβ1‐subunits in distal, but not proximal colon.Grant Funding Source: West Virgina University Intramural award

Maternal protein restriction during pregnancy affects gene expression and immunolocalization of intestinal nutrient transporters in rats

Intrauterine dietary restriction may cause changes in the functioning of offspring organs and systems later in life, an effect known as fetal programming. The present study evaluated mRNA abundance and immunolocalization of nutrient transporters as well as enterocytes proliferation in the proximal, median and distal segments of small intestine of rats born to protein-restricted dams. Pregnant rats were fed hypoproteic (6% protein) or control (17% protein) diets, and offspring rats were evaluated at 3 and 16 weeks of age. The presence of SGLT1 (sodium-glucose co-transporter 1), GLUT2 (glucose transporter 2), PEPT1 (peptide transporter 1) and the intestinal proliferation were evaluated by immunohistochemical techniques and the abundance of specific mRNA for SGLT1, GLUT2 and PEPT1 was assessed by the real-time PCR technique. Rats born to protein-restricted dams showed higher cell proliferation in all intestinal segments and higher gene expression of SGLT1 and PEPT1 in the duodenum. Moreover, in adult animals born to protein-restricted dams the immunoreactivity of SGLT1, GLUT2 and PEPT1 in the duodenum was more intense than in control rats. Taken together, the results indicate that changes in the small intestine observed in adulthood can be programmed during the gestation. In addition, they show that this response is caused by both up-regulation in transporter gene expression, a specific adaptation mechanism, and intestinal proliferation, an unspecific adaptation mechanism.

HIF-1 and SKN-1 Coordinate the Transcriptional Response to Hydrogen Sulfide in Caenorhabditis elegans

Hydrogen sulfide (H2S) has dramatic physiological effects on animals that are associated with improved survival. C. elegans grown in H2S are long-lived and thermotolerant. To identify mechanisms by which adaptation to H2S effects physiological functions, we have measured transcriptional responses to H2S exposure. Using microarray analysis we observe rapid changes in the abundance of specific mRNAs. The number and magnitude of transcriptional changes increased with the duration of H2S exposure. Functional annotation suggests that genes associated with protein homeostasis are upregulated upon prolonged exposure to H2S. Previous work has shown that the hypoxia-inducible transcription factor, HIF-1, is required for survival in H2S. In fact, we show that hif-1 is required for most, if not all, early transcriptional changes in H2S. Moreover, our data demonstrate that SKN-1, the C. elegans homologue of NRF2, also contributes to H2S-dependent changes in transcription. We show that these results are functionally important, as skn-1 is essential to survive exposure to H2S. Our results suggest a model in which HIF-1 and SKN-1 coordinate a broad transcriptional response to H2S that culminates in a global reorganization of protein homeostasis networks.

Differential programming of p53-deficient embryonic cells during rotenone block

Mitochondrial dysfunction has been implicated in chemical toxicities. The present study used an in vitro model to investigate the differential expression of metabolic pathways during cellular stress in p53-efficient embryonic fibroblasts compared to p53-deficient cells. These cell lines differed with respect to NADH/NAD + balance. This ratio constitutes a driving force for NAD- and NADH-dependent reactions and is inversed upon exposure to Rotenone (complex I inhibitor). Rotenone perturbed the structure of the elongated fibrillar tubulin network and decreased mRNA expression of tubulin genes both suggesting reprogramming and reorganization of the cytoskeleton in both cell lines. These changes were reflected in the abundance of specific mRNA and microRNA (miRNA) species as determined from genome-based analysis. Changes in mRNA and miRNA expression profiles reflected differences in energy utilizing pathways, consistent with the notion that the p53 pathway influences the cellular response to mitochondrial dysfunction and that at least some control may be embedded within specific mRNA/miRNA networks in embryonic cells.

Proinflammatory Cytokine TNF‐α Inhibits Na+‐glutamine Cotransport in Intestinal Epithelial Cell line (IEC‐6)

Mucosal Na+-glutamine (Gln) cotransport is critical in absorbing exogenous Gln that the body needs during extreme stress (e.g. infections and trauma). Although stress has been shown to inhibit Gln synthesis, it is not known whether stress also inhibits Gln absorption. Thus, the aim of this study was to determine whether TNF-α, the major proinflammatory cytokine is produced during infection would inhibit Gln cotransport in rat intestinal epithelial (IEC-6) cells. [3H]-Gln uptake was measured in 10-day post-confluent normal and TNF-α (25 ng/ml) treated IEC-6 cells, grown in 6-well plates. Na+/K+-ATPase activity, and B0AT1 specific mRNA abundance and protein expression were measured by qRT-PCR and western blot analyses, respectively. Na+-Gln cotransport is present in IEC-6 cells. TNF-α exposure (24 hrs) inhibited Na+-Gln cotransport by 75% (1.9 ± 0.1 vs 0.5 ± 0.01 nmol/mg protein • 2 min). Kinetic studies revealed that TNF-α inhibited Na+-Gln cotransport by reducing the affinity for Gln (Km: normal vs TNF-α = 3.5 ± 0.3 vs 8.5 ± 1.0 mM). TNF-α did not alter either B0AT1 specific mRNA abundance and protein expression or Na+/K+-ATPase activity. We concluded from these results that, in addition to inhibiting Gln synthesis, extreme stress also inhibits exogenous Gln absorption. We speculate that TNF-α might regulate Na+-Gln cotransport mediated via B0AT1 by second messenger mediated signal transduction process(es).

Tight junction proteins in zebrafish ovarian follicles: Stage specific mRNA abundance and response to 17β-estradiol, human chorionic gonadotropin, and maturation inducing hormone

In vertebrate epithelia, the tight junction (TJ) complex plays an important role in the regulation of paracellular permeability and contributes to mechanical stability. Using zebrafish, this study examined the possibility that TJ protein ‘machinery’ may contribute to the complex process of ovarian follicle development in fishes and be responsive to key endocrine factors that assist in the regulation of this event. Transcript encoding for 18 zebrafish claudin ( cldn) and 2 occludin ( ocln) orthologs were widely distributed in zebrafish tissues. All orthologs were detected in the ovary, albeit at varying levels of abundance. Using occludin as a marker, TJs localized to the periphery of ovarian follicles, corresponding to the position of granulosa and theca cells. Of the 20 orthologs examined, mRNA encoding for cldn g, d, and ocln were most abundant in whole ovary. As follicles transitioned from pre-vitellogenic to mid-/late-stage vitellogenic status, mRNA encoding for cldn a, b, d, g, h and 19 significantly declined. Transcript encoding for other orthologs (i.e. cldn c, e, f, i, j, 2, 7, 8, 10, 11, 12, 15, ocln and ocln b) did not significantly alter during follicle development. Exogenous 17β-estradiol had little effect on TJ machinery in pre-vitellogenic follicles, but reduced the abundance of cldn h and 10 in mid- to late-stage vitellogenic follicles. In mid-vitellogenic follicles human chorionic gonadotropin increased cldn d and g mRNA abundance, whereas maturation inducing hormone reduced cldn h and 19 mRNA abundance. The data supports a role for the endocrine regulation of TJ proteins in ovarian follicle development.

Rho-Mediated Regulation of TGF-β1– and FGF-2–Induced Activation of Corneal Stromal Keratocytes

To investigate the role of Rho GTPase signaling in FGF-2- and TGF-beta1-induced activation of corneal keratocytes. Keratocytes isolated from rabbit corneal stroma and plated in a serum-free medium were treated with FGF-2/heparin or TGF-beta1 in the presence or absence of Rho inhibitor (C3 exoenzyme) or ROCK (Rho kinase) inhibitor (Y27632). Specific phenotypic changes were analyzed by immunocytochemistry and Western blot analysis, and the relative abundance of specific mRNAs was estimated by quantitative RT-PCR. TGF-beta1-induced expression of alpha-SMA and transcription of alpha-SMA mRNA in activated keratocytes were reduced by Rho or ROCK inhibition during the activation. In nonactivated keratocytes, the expression of alpha3(IV) collagen was downregulated by Rho-inhibition. TGF-beta1- or FGF-2-induced downregulation of the expression of alpha3(IV) collagen and its mRNA was not significantly altered by Rho or ROCK inhibition. TGF-beta1- and FGF-2-induced decreases in cell-associated and secreted KS, and lumican mRNA levels were prevented by Rho or ROCK inhibition. However, FGF-2-induced decreases in keratocan mRNA levels were prevented by Rho inhibition but not by ROCK inhibition. Whereas Rho inhibition downregulated both TGF-beta1- and FGF-2-induced tenascin-C expression, ROCK inhibition was found to downregulate only TGF-beta1-induced expression. Rho signaling has a significant role in the activation of keratocytes. Rho, via ROCK-independent and/or -dependent pathways differentially regulates the TGF-beta1-induced expression of alpha-SMA and TGF-beta1- and FGF-2-induced de novo expression of tenascin-C and the downregulation of alpha3(IV) collagen and KSPGs, lumican and keratocan.

Superoxide-Dependent Cathepsin Activation Is Associated with Hypertensive Myocardial Remodeling and Represents a Target for Angiotensin II Type 1 Receptor Blocker Treatment

The elastolytic activity of cathepsins in the myocardium is implicated in hypertensive heart failure (HF). Given that reactive oxygen species are also implicated in protease activation associated with cardiac remodeling, we examined the role of the reactive oxygen species-induced cathepsin activation system in cardiac remodeling during the development of hypertensive HF. Dahl salt-sensitive hypertensive rats maintained on a high-salt diet were treated with vehicle, the cathepsin inhibitor E64d, or the angiotensin receptor blocker olmesartan from 12 to 19 weeks of age. Cathepsin expression and activity were increased in the left ventricle of HF rats; olmesartan inhibited these effects, restored the balance between elastin and collagen in the left ventricle, and suppressed degradation of the elastic lamina of coronary arteries of HF rats. Furthermore, olmesartan inhibited up-regulation of NADPH oxidase subunits and activity as well as superoxide generation. These effects of olmesartan were mimicked by E64d and were accompanied by amelioration of cardiac fibrosis. Finally, olmesartan and apocynin reduced angiotensin II-induced increases in cathepsin mRNA and protein levels in cultured rat neonatal cardiac myocytes. These data suggest that cathepsins likely trigger and promote cardiac remodeling and that blocking the angiotensin II type 1 receptor attenuates cathepsin expression and activity by inhibiting the production of superoxide by NADPH oxidase, thereby attenuating cardiac remodeling and dysfunction.

Transcriptome analysis of acclimatory responses to thermal stress in Antarctic algae

A customized cDNA chip analysis provided the relative expression profiling of 1439 ESTs of Chaetoceros neogracile in culture environments maintained between 4 and 10°C. Among the 1439 probes, 21.5% were differentially regulated (⩾2-fold) by the temperature upshift within three days. Up-regulation was more prominent among cytoprotective genes, whereas down-regulation was featured in photosynthetic genes. A third of the differentially expressed genes had an unknown function or no similarity to known genes, highlighting their potential importance as a resource to identify key players in the acclimation response of polar algae under thermal stress. Our transcriptome analysis also revealed novel aspects of temperature-responsive, coordinated changes in the abundance of specific mRNAs, along with the rapid establishment of molecular homeostasis in polar algae. Unexpectedly, a small set of genes encoding fucoxanthin chlorophyll a/c-binding proteins were rapidly up-regulated by thermal stress, implying that they have different roles other than light harvesting.

Transient changes in the expression pattern of key enzymes for bile acid synthesis during rat liver regeneration

Changes in the expression patterns of genes involved in bile acid (BA) synthesis were investigated during rat liver regeneration that follows two-thirds partial hepatectomy. BAs in bile were measured by GC-MS and the absolute and relative abundance of specific mRNAs in the liver by RT-real-time quantitative PCR. Cyclin E mRNA, used as an indicator of liver cell proliferation, peaked at day 1. The levels of mRNA of α-fetoprotein transcription factor (FTF) and small heterodimer partner (SHP) were first reduced (day 1) and then (days 2–3) increased, when those of farnesoid X receptor (FXR) were also transiently enhanced. The early (day 1) up-regulation of Cyp7a1, and Cyp8b1, together with the down-regulation of Cyp27, was consistent with an increased proportion of cholic acid versus chenodeoxycholic acid and a progressive recovery in total BAs secretion. The transient appearance of flat BAs ( allo-BAs plus Δ 4-unsaturated-BAs) during rat liver regeneration was probably due to the changes in the expression ratio of steroid 5α- versus 5β-reductase. Both were first (day 1) down-regulated and then up-regulated (5α-reductase more than 5β-reductase). In conclusion, changes in the expression patterns of nuclear receptors and enzymes involved in BA synthesis are consistent with the transient modifications that occur in BA pool during rat liver regeneration.

Messenger RNA stockpile of cyclin B, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor receptor Ib, and p53 in the rainbow trout oocyte in relation with developmental competence.

In the present study, correlations between the oocyte messenger RNA (mRNA) stockpile of Cyclin B, insulin-like growth factor I (IGF-I), insulin-like growth factor (IGF-II), insulin-like growth factor receptor Ib (IGFR Ib), and p53 transcripts and the developmental competence of the oocyte were studied. For this purpose, post-ovulatory ageing was used as a tool to generate oocytes of varying developmental competence. Mature female rainbow trout were held at 12 degrees C and periodically checked for ovulation. Oocytes were collected from each female at ovulation and 5, 14, 21 days later. For each collected egg batch, the abundance of several mRNAs in the oocyte was analyzed by real-time PCR and embryo development was monitored after fertilization. Egg quality was estimated not only through embryonic survival but also by studying the occurrence of specific morphological abnormalities. The present study showed that oocyte post-ovulatory ageing was associated with variations of the relative abundance of several studied transcripts within the oocyte. In addition, the abundance of specific mRNAs could be correlated with either the embryonic survival or the occurrence of malformations. Thus, the abundance of IGFR Ib and Cyclin B transcripts in the oocyte was correlated with the occurrence of morphological abnormalities observed at yolk-sac resorption (negatively for IGFR Ib and positively for Cyclin B), while the maternal stockpile of IGF-I, IGF-II, and IGFR Ib mRNAs was positively correlated with embryonic survival.