RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Identification of protein-coding and non-coding RNA expression profiles in CD34(+) and in stromal cells in refractory anemia with ringed sideroblasts

Background: Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods: In this study, gene expression profiles of CD34(+) cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results: In CD34(+) cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value <= 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value <= 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions: These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34(+) cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

The expression of messenger RNAs coding for growth factors, their receptors, and eph-class receptor tyrosine kinases in normal and ototoxically damaged chick cochleae

Messenger RNAs coding for growth factors and receptor tyrosine kinases were measured by quantitative competitive and by semi-quantitative reverse-transcription polymerase chain reaction in whole and dissected chick inner ears. The fibroblast growth factor (FGF) receptor 1 chick embryonic kinase (CEK) 1 was expressed in all structures examined (otocyst, hatchling whole cochlea, cochlear nerve ganglion, and cochlear and vestibular sensory epithelia), although slightly more heavily in the otocyst. The related fibroblast growth factor receptors CEK 2 and 3 were preferentially expressed in the nerve ganglion and in the vestibular sensory epithelium, respectively. FGF 1 mRNA was low in early development, increasing to mature levels at around embryonic age 11 days, while FGF2, mRNA was expressed at constant levels at all ages. In response to ototoxic damage, FGF1 mRNA levels were increased in the early damaged cochlear sensory epithelium. Immunohistochemistry for CEK1 showed that normal hair cells expressed the receptor heavily on the hair cell stereocilia, while with early damage, CEK1 came to be expressed heavily on the apical surfaces of the supporting cells. In normal chicks, the CEK4 and CEK8 eph-class receptor tyrosine kinases were expressed relatively heavily by the cochlear nerve ganglion, and CEK10 was expressed relatively heavily by the cochlear hair cell sensory epithelium. The results suggest that the FGF system may be involved in the response of the cochlear epithelium to ototoxic damage. The eph-class receptor tyrosine kinase CEK10 may be involved in cell interactions in the cochlear sensory epithelium, while CEK4 and CEK8 may play a role in the cochlear innervation.

Rapid isolation of high-quality RNA from symbiotic dinoflagellates

This report details a reliable and efficient RNA extraction protocol for the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal (Gymnodiniales, Dinophyceae). The method typically gives yields of 500 mu g total RNA from 0.4 g wet weight of algae, and, in comparison to current protocols, it is technically simple and less time consuming. This method isolates high-quality, intact RNA from in vine cultured as well as host-isolated cells, as demonstrated by spectrophotometry, gel electrophoresis, and northern analysis. The total RNA obtained was suitable for reverse transcription and PCR amplification of Symbiodinium cDNAs. We have successfully applied our method to isolate total RNA from a different dinoflagellate, Amphidinium carterae Hulburt (Gymnodiniales, Dinophyceae), found in symbiotic association with marine invertebrates.

Distinguishing species and populations of Rhipicephaline ticks with its 2 ribosomal RNA

Most populations and some species of ticks of the genera Boophilus (5 spp.) and Rhipicephalus (ca. 75 spp.) cannot be distinguished phenotypically. Moreover, there is doubt about the validity of species in these genera. I studied the entire second internal transcribed spacer (ITS 2) rRNA of 16 populations of rhipicephaline ticks to address these problems: Boophilus,microplus from Australia, Kenya, South Africa and Brazil (4 populations); Boophilus decoloratus from Kenya; Rhipicephalus appendiculatus from Kenya, Zimbabwe and Zambia (7 populations); Rhipicephalus zambesiensis from Zimbabwe (3 populations); and Rhipicephalus evertsi from Kenya. Each of the 16 populations had a unique ITS 2, but most of the nucleotide variation occurred among species and genera. ITS 2 rRNA can be used to distinguish the populations and species of Boophilus and Rhipicephalus studied here. Little support was found for the hypothesis that B. microplus from Australia and South Africa are different species. ITS 2 appears useful for phylogenetic inference in the Rhipicephalinae because in genetic distance, maximum likelihood, and maximum parsimony analyses, most branches leading to species had >95% bootstrap support. Rhipicephalus appendiculatus and R, zambeziensis are closely related, yet their ITS 2 sequences could be distinguished unambiguously. This lends weight to a previous proposal that Rhipicephalus sanguineus and Rhipicephalus turanicus, and Rhipicephalus pumlilio and Rhipicephalus camicasi, respectively, are conspecific, because each of these pairs of species had identical sequences for ca. 250 bp of ITS 2 rRNA.

Reverse transcription of a naturally occurring nonretroviral RNA produces a precise deletion in the majority of its cDNA products

A precise, reproducible deletion made during in vitro reverse transcription of RNA2 from the icosahedral positive-stranded Helicoverpa armigera stunt virus (Tetraviridae) is described. The deletion, located between two hexamer repeats, is a 50-base sequence that includes one copy of the hexamer repeat. Only the Moloney murine leukemia virus reverse transcriptase and its derivative Superscript I, carrying a deletion of the carboxy-terminal RNase H region, showed this response, indicating a template-switching mechanism different from one proposed that involves a RNase H-dependent strand transfer, Superscript II, however, which carries point mutations to reduce RNase H activity, does not cause a deletion. A possible mechanism involves the enzyme pausing at the 3' side of a stem-loop structure and the 3' end of the nascent DNA strand separating from the template and reannealing to the upstream hexamer repeat.

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Two small RNAs regulate the timing of Caenorhabditis elegans development(1,2). Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA(1,3,4), and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA 2. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs1,2,5,6. Here we have detected let-7 RNAs of similar to 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.

Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA - Crystal structure of an oligomeric complex of yeast SmF

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.

Influence of Lactic Acid on Endogenous and Viral RNA-Induced Immune Mediator Production by Vaginal Epithelial Cells

OBJECTIVE: To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. METHODS: The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid: cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1 beta, IL-6, IL-8, IL-23, transforming growth factor (TGF)-beta and secretory leukocyte protease inhibitor. RESULTS: Vaginal epithelial cells spontaneously released IL-1 beta (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-beta (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-beta production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid: cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid: cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1 beta (96.2 pg/mL). The poly (inosinic acid: cytidylic acid)-mediated lactic acid effect on IL-1 beta and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. CONCLUSION: Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses. (Obstet Gynecol 2011;118:840-6) DOI: 10.1097/AOG.0b013e31822da9e9

Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GN in early embryogenesis?

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages, In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose-response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40-50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GK. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription-PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.

Association of low sodium-iodide symporter messenger ribonucleic acid expression in malignant thyroid nodules with increased intracellular protein staining

Context: The expression of sodium iodide symporter (NIS) is required for iodide uptake in thyroid cells. Benign and malignant thyroid tumors have low iodide uptake. However, previous studies by RT-PCR or immunohistochemistry have shown divergent results of NIS expression in these nodules. Objective: The objective of the study was to investigate NIS mRNA transcript levels, compare with NIS and TSH receptor proteins expression, and localize the NIS protein in thyroid nodules samples and their surrounding nonnodular tissues (controls). Design: NIS mRNA levels, quantified by real-time RT-PCR, and NIS and TSH receptor proteins, evaluated by immunohistochemistry, were examined in surgical specimens of 12 benign and 13 malignant nodules and control samples. Results: When compared with controls, 83.3% of the benign and 100% of the malignant nodules had significantly lower NIS gene expression. Conversely, 66.7% of the benign and 100% of malignant nodules had stronger intracellular NIS immunostaining than controls. Low gene expression associated with strong intracellular immunostaining was most frequently detected in malignant (100%) than benign nodules (50%; P = 0.005). NIS protein was located at the basolateral membrane in 24% of the control samples, 8.3% of the benign, and 15.4% of the malignant nodules. The percentage of benign nodules with strong TSH receptor positivity (41.6%) was higher than malignant (7.7%). Conclusion: We confirmed that reduced NIS mRNA expression in thyroid malignant nodules is associated with strong intracellular protein staining and may be related to the inability of the NIS protein to migrate to the cellular basolateral membrane. These results may explain the low iodide uptake of malignant nodules.

Inhibition of Expression of HTLV-1 Structural Genes Mediated by Short Hairpin RNA In Vitro

Background: Human T-lymphotropic virus 1 (HTLV-1) is associated with the T-cell malignancy known as adult T-cell leukemia! lymphoma (ATLL) and with a disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, the treatment of these diseases is based on symptom relief. RNA interference (RNAi) technology has been described as an efficient mechanism for development of new therapeutic methods. Thus, the aim of this study was to evaluate the inhibition of HTLV-1 structural proteins using short hairpin RNAs (shRNAs) expressed by non-viral vectors. Materials and Methods: Reporter plasmids that express enhanced green fluorescent protein-Gag (EGFP-Gag) and EGFP-Env fusion proteins and vectors that express shRNAs corresponding to the HTLV-1 gag and env genes were constructed. shRNA vectors and reporter plasmids were simultaneously transfected into HEK 293 cells. Results: Fluorescence microscopy, flow cytometry and real-time PCR showed that shRNAs were effective in inhibiting the fusion proteins. Conclusion: These shRNAs are effective against the expression of structural genes and may provide an approach to the development of new therapeutic agents.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA(1,2). Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in E mu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc- overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap- dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site ( IRES)- dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic- specific expression of the endogenous IRES- dependent form of Cdk11 ( also known as Cdc21 and PITSLRE)(3-5), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in E mu-Myc/+ mice. When accurate translational control is re- established in E mu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post- genomic level.