Genome mapping and expression analyses of human intronic noncoding RNAs reveal tissue-specific patterns and enrichment in genes related to regulation of transcription

Abstract Background RNAs transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression. However, the complement of human genes in which introns are transcribed, and the number of intronic transcriptional units and their tissue expression patterns are not known. Results A survey of mRNA and EST public databases revealed more than 55,000 totally intronic noncoding (TIN) RNAs transcribed from the introns of 74% of all unique RefSeq genes. Guided by this information, we designed an oligoarray platform containing sense and antisense probes for each of 7,135 randomly selected TIN transcripts plus the corresponding protein-coding genes. We identified exonic and intronic tissue-specific expression signatures for human liver, prostate and kidney. The most highly expressed antisense TIN RNAs were transcribed from introns of protein-coding genes significantly enriched (p = 0.002 to 0.022) in the 'Regulation of transcription' Gene Ontology category. RNA polymerase II inhibition resulted in increased expression of a fraction of intronic RNAs in cell cultures, suggesting that other RNA polymerases may be involved in their biosynthesis. Members of a subset of intronic and protein-coding signatures transcribed from the same genomic loci have correlated expression patterns, suggesting that intronic RNAs regulate the abundance or the pattern of exon usage in protein-coding messages. Conclusion We have identified diverse intronic RNA expression patterns, pointing to distinct regulatory roles. This gene-oriented approach, using a combined intron-exon oligoarray, should permit further comparative analysis of intronic transcription under various physiological and pathological conditions, thus advancing current knowledge about the biological functions of these noncoding RNAs.

Characterization of a Novel Oral Glucocorticoid System and Its Possible Role in Disease

Synthetic corticosteroids are used widely for the treatment of a variety of diseases of the mouth. However, little is known as to whether the oral mucosa is able to modulate the local concentration of active corticosteroids or to produce steroids de novo. This has important clinical implications, because tissue-specific regulation of glucocorticoids is a key determinant of the clinical efficacy of these drugs. In the present study, we show that oral fibroblasts and keratinocytes expressed ACTH receptor (MC2R), glucocorticoid receptor (GR), and 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs). Unlike keratinocytes, fibroblasts lacked 11 beta-HSD2 and could not effectively deactivate exogenously administered cortisol. However, both cell types were able not only to activate cortisone into the active form cortisol, but also to synthesize cortisol de novo following stimulation with ACTH. 11 beta-HSD2, the enzyme controlling cortisol deactivation, exhibited different patterns of expression in normal (squamous epithelium and salivary glands) and diseased oral mucosa (squamous cell carcinoma and mucoepidermoid carcinoma). Blocking of endogenous cortisol catabolism in keratinocytes with the 11 beta-HSD2 inhibitor 18 beta-glycyrrhetinic acid mimicked the effect of exogenous administration of hydrocortisone and partially prevented the detrimental effects induced by pemphigus vulgaris sera. Analysis of the data demonstrates that a novel, non-adrenal glucocorticoid system is present in the oral mucosa that may play an important role in disease.

Experimental Basis and New Insights for Cell Therapy in Chronic Obstructive Pulmonary Disease

Chronic Obstructive Pulmonary Disease (COPD) can be briefly described as air flow limitation and chronic dyspnea associated to an inflammatory response of the respiratory tract to noxious particles and gases. Its main feature is the obstruction of airflow and consequent chronic dyspnea. Despite recent advances, and the development of new therapeutic, medical and clinical approaches, a curative therapy is yet to be achieved. Therapies involving the use of tissue-specific or donor derived cells present a promising alternative in the treatment of degenerative diseases and injuries. Recent studies demonstrate that mesenchymal stem cells have the capacity to modulate immune responses in acute lung injury and pulmonary fibrosis in animal models, as well as in human patients. Due to these aspects, different groups raised the possibility that the stem cells from different sources, such as those found in bone marrow or adipose tissue, could act preventing the emphysematous lesion progression. In this paper, it is proposed a review of the current state of the art and future perspectives on the use of cell therapy in obstructive lung diseases.

Fhos encodes a Drosophila Formin-Like Protein participating in autophagic programmed cell death

Larval tissues undergo programmed cell death (PCD) during Drosophila metamorphosis. PCD is triggered in a stage and tissue-specific fashion in response to ecdysone pulses. The understanding of how ecdysone induces the stage and tissue-specificity of cell death remains obscure. Several steroid-regulated primary response genes have been shown to act as key regulators of cellular responses to ecdysone by inducing a cascade of transcriptional regulation of late responsive genes. In this article, the authors identify Fhos as a gene that is required for Drosophila larval salivary gland destruction. Animals with a P-element mutation in Fhos possess persistent larval salivary glands, and precise excisions of this P-element insertion resulted in reversion of this salivary gland mutant phenotype. Fhos encodes the Drosophila homolog of mammalian Formin Fhos. Fhos is differentially transcribed during development and responds to ecdysone in a method that is similar to other cell death genes. Similarly to what has been shown for its mammalian counterpart, FHOS protein is translocated to the nucleus at later stages of cell death. Fhos mutants posses disrupted actin cytoskeleton dynamics in persistent salivary glands. Together, our data indicate that Fhos is a new ecdysone-regulated gene that is crucial for changes in the actin cytoskeleton during salivary gland elimination in Drosophila. genesis 50:672684, 2012. (c) 2012 Wiley Periodicals, Inc.

Prion potency in stem cells biology

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

Avian coronavirus Spike Glycoprotein Ectodomain Shows a Low Codon Adaptation to Gallus gallus with Virus-Exclusive Codons in Strategic Amino Acids Positions

This is a study on the Avian coronavirus IBV and chicken host-relationship from the codon usage point of view based on fifty-nine non-redundant IBV S1 sequences (nt 1-507) from strains detected worldwide and chicken tissue-specific protein genes sequences from IBV-replicating sites. The effective number of codons (ENC) values ranged from 36 to 47.8, indicating a high-to-moderate codon usage bias. The highest IBV codon adaptation index (CAI) value was 0.7, indicating a distant virus versus host synonymous codons usage. The ENC x GC3 % curve indicates that both mutational pressure and natural selection are the driving forces on codon usage pattern in S1. The low CAI values agree with a low S protein expression and considering that S protein is a determinant for attachment and neutralization, this could be a further mechanism besides mRNA transcription attenuation for a low expression of this protein leading to an immune camouflage.

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 degrees C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K-m = 37.53 mM S-1), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K-i = 0.196 nM), indicating that this protease is a potential target for pest control. (C) 2011 Elsevier Ltd. All rights reserved.

Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT. Hum Mutat 33: 949-959, 2012. (C) 2012 Wiley Periodicals, Inc.

Assessment of the role of transcript for GATA-4 as a marker of unfavorable outcome in human adrenocortical neoplasms

Abstract Background Malignant neoplasia of the adrenal cortex is usually associated with very poor prognosis. When adrenocortical neoplasms are diagnosed in the early stages, distinction between carcinoma and adenoma can be very difficult to accomplish, since there is yet no reliable marker to predict tumor recurrence or dissemination. GATA transcription factors play an essential role in the developmental control of cell fate, cell proliferation and differentiation, organ morphogenesis, and tissue-specific gene expression. Normal mouse adrenal cortex expresses GATA-6 while its malignant counterpart only expresses GATA-4. The goal of the present study was to assess whether this reciprocal change in the expression of GATA factors might be relevant for predicting the prognosis of human adrenocortical neoplasms. Since human adrenal cortices express luteinizing hormone (LH/hCG) receptor and the gonadotropins are known to up-regulate GATA-4 in gonadal tumor cell lines, we also studied the expression of LH/hCG receptor. Methods We conducted a study on 13 non-metastasizing (NM) and 10 metastasizing/recurrent (MR) tumors obtained from a group of twenty-two adult and pediatric patients. The expression of GATA-4, GATA-6, and LH/hCG receptor (LHR) in normal and tumoral human adrenal cortices was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR) complemented by dot blot hybridization. Results Messenger RNA for GATA-6 was detected in normal adrenal tissue, as well as in the totality of NM and MR tumors. GATA-4, by its turn, was detected in normal adrenal tissue, in 11 out of 13 NM tumors, and in 9 of the 10 MR tumors, with larger amounts of mRNA found among those presenting aggressive clinical behavior. Transcripts for LH receptor were observed both in normal tissue and neoplasms. A more intense LHR transcript accumulation was observed on those tumors with better clinical outcome. Conclusion Our data suggest that the expression of GATA-6 in human adrenal cortex is not affected by tumorigenesis. GATA-4 expression is more abundant in MR tumors, while NM tumors express more intensely LHR. Further studies with larger cohorts are needed to test whether relative expression levels of LHR or GATA-4 might be used as prognosis predictors.

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Abstract Background Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination. Methods Uteri from mice in all phases of the estrous cycle, and animals subjected to ovariectomy and hormone replacement were collected for immunoperoxidase staining for versican, as well as PCR and quantitative Real Time PCR. Results In diestrus and proestrus, VER was exclusively expressed in the endometrial stroma. In estrus and metaestrus, VER was present in both endometrial stroma and myometrium. In OVX mice, VER immunoreaction was abolished in all uterine tissues. VER expression was restored by E2, MPA and E2+MPA treatments. Real Time PCR analysis showed that VER expression increases considerably in the MPA-treated group. Analysis of mRNA identified isoforms V0, V1 and V3 in the mouse uterus. Conclusion These results show that the expression of versican in uterine tissues is modulated by ovarian steroid hormones, in a tissue-specific manner. VER is induced in the myometrium exclusively by E2, whereas MPA induces VER deposition only in the endometrial stroma.

Bladder expression of CD cell surface antigens and cell-type-specific transcriptomes

Many cell types have no known functional attributes. In the bladder and prostate, basal epithelial and stromal cells appear similar in cytomorphology and share several cell surface markers. Their total gene expression (transcriptome) should provide a clear measure of the extent to which they are alike functionally. Since urologic stromal cells are known to mediate organ-specific tissue formation, these cells in cancers might exhibit aberrant gene expression affecting their function. For transcriptomes, cluster designation (CD) antigens have been identified for cell sorting. The sorted cell populations can be analyzed by DNA microarrays. Various bladder cell types have unique complements of CD molecules. CD9(+) urothelial, CD104(+) basal and CD13(+) stromal cells of the lamina propria were therefore analyzed, as were CD9(+) cancer and CD13(+) cancer-associated stromal cells. The transcriptome datasets were compared by principal components analysis for relatedness between cell types; those with similarity in gene expression indicated similar function. Although bladder and prostate basal cells shared CD markers such as CD104, CD44 and CD49f, they differed in overall gene expression. Basal cells also lacked stem cell gene expression. The bladder luminal and stromal transcriptomes were distinct from their prostate counterparts. In bladder cancer, not only the urothelial but also the stromal cells showed gene expression alteration. The cancer process in both might thus involve defective stromal signaling. These cell-type transcriptomes provide a means to monitor in vitro models in which various CD-isolated cell types can be combined to study bladder differentiation and bladder tumor development based on cell-cell interaction.

Fluctuation of Anti-Endothelial Cell Antibody Titers in Mixed Connective Tissue Disease

Background: Antibodies directed against endothelial cell surface antigens have been described in many disorders and have been associated with disease activity. Since the most prominent histopathologic feature in mixed connective tissue disease (MCTD) is the widespread and unique proliferative vascular lesion, our aim was to evaluate the frequency of anti-endothelial cell antibodies (AECA) in this condition. Objectives: To evaluate the frequency of AECA in this disease and assess its clinical and laboratory associations. Methods: Seventy-three sera from 35 patients with MCTD (Kasukawa's criteria), collected during a 7 year period, were tested for immunoglobulins G and M (IgG and IgM) AECA by cellular ELISA, using HUVEC (human umbilical vein endothelial cells). Sera from 37 patients with systemic lupus erythematosus (SLE), 22 with systemic sclerosis (SSc) and 36 sera from normal healthy individuals were used as controls. A cellular ELISA using HeLa cells was also performed as a laboratory control method. Results: IgG-AECA was detected in 77% of MCTD patients, 54% of SLE patients, 36% of SSc patients and 6% of normal controls. In MCTD, IgG-AECA was associated with vasculitic manifestations, disease activity and lymphopenia, and was also a predictor of constant disease activity. Immunosuppressive drugs were shown to reduce IgG-AECA titers. Since antibodies directed to HeLa cell surface were negative, AECA was apparently unrelated to common epitopes present on epithelial cell lines. Conclusions: AECA are present in a large proportion of patients with MCTD and these antibodies decrease after immunosuppressive treatment. IMAJ 2012; 14:84-87

Early detection of doxorubicin myocardial injury by ultrasonic tissue characterization in an experimental animal model

In the clinical setting, the early detection of myocardial injury induced by doxorubicin (DXR) is still considered a challenge. To assess whether ultrasonic tissue characterization (UTC) can identify early DXR-related myocardial lesions and their correlation with collagen myocardial percentages, we studied 60 rats at basal status and prospectively after 2mg/Kg/week DXR endovenous infusion. Echocardiographic examinations were conducted at baseline and at 8,10,12,14 and 16 mg/Kg DXR cumulative dose. The left ventricle ejection fraction (LVEF), shortening fraction (SF), and the UTC indices: corrected coefficient of integrated backscatter (IBS) (tissue IBS intensity/phantom IBS intensity) (CC-IBS) and the cyclic variation magnitude of this intensity curve (MCV) were measured. The variation of each parameter of study through DXR dose was expressed by the average and standard error at specific DXR dosages and those at baseline. The collagen percent (%) was calculated in six control group animals and 24 DXR group animals. CC-IBS increased (1.29 +/- 0.27 x 1.1 +/- 0.26-basal; p=0.005) and MCV decreased (9.1 +/- 2.8 x 11.02 +/- 2.6-basal; p=0.006) from 8 mg/Kg to 16mg/Kg DXR. LVEF presented only a slight but significant decrease (80.4 +/- 6.9% x 85.3 +/- 6.9%-basal, p=0.005) from 8 mg/Kg to 16 mg/Kg DXR. CC-IBS was 72.2% sensitive and 83.3% specific to detect collagen deposition of 4.24%(AUC=0.76). LVEF was not accurate to detect initial collagen deposition (AUC=0.54). In conclusion: UTC was able to early identify the DXR myocardial lesion when compared to LVEF, showing good accuracy to detect the initial collagen deposition in this experimental animal model.

Relationship between B-Cell-specific moloney murine leukemia virus integration site 1 (BMI-1) and homologous recombination regulatory genes in invasive ductal breast carcinomas

B-cell-specific Moloney murine leukemia virus integration site 1 (Bmi-1) is a Polycomb group protein that is able to induce telomerase activity, enabling the immortalization of epithelial cells. Immortalized cells are more susceptible to double-strand breaks (DSB), which are subsequently repaired by homologous recombination (HR). BRCA1 is among the HR regulatory genes involved in the response to DNA damage associated with the RAD51 protein, which accumulates in DNA damage foci after signaling H2AX, another important marker of DNA damage. Topoisomerase III beta (topoIII beta) removes HR intermediates before chromosomal segregation, preventing damage to cellular DNA structure. In breast carcinomas positive for BMI-1 the role of proteins involved in HR remains to be investigated. The aim of this study was to evaluate the association between BMI-1 and homologous recombination proteins. Using tissue microarrays containing 239 cases of primary breast tumors, the expression of Bmi-1, BRCA-1, H2AX, Rad51, p53, Ki-67, topoIII beta, estrogen receptors (ER), progesterone receptors (PR), and HER-2 was analyzed by immunohistochemistry. We observed high Bmi-1 expression in 66 cases (27.6%). Immunohistochemical overexpression of BMI-1 was related to ER (p=0.004), PR (p<0.001), Ki-67 (p<0.001), p53 (p=0.003), BRCA1 (p=0.003), H2AX (p=0.024) and topoIII beta (p<0,001). Our results show a relationship between the expression of BMI-1 and HR regulatory genes, suggesting that Bmi-1 overexpression might be an important event in HR regulation. However, further studies are necessary to understand the mechanisms in which Bmi-1 could regulate HR pathways in invasive ductal breast carcinomas.

Expression of cancer-testis antigens MAGE-A4 and MAGE-C1 in oral squamous cell carcinoma

Background Tumor markers are genes or their products expressed exclusively or preferentially in tumor cells and cancer-testis antigens (CTAs) form a group of genes with a typical expression pattern expressed in a variety of malignant neoplasms. CTAs are considered potential targets for cancer vaccines. It is possible that the CTA MAGE-A4 (melanoma antigen) and MAGE-C1 are expressed in carcinoma of the oral cavity and are related with survival. Methods This study involved immunohistochemical analysis of 23 patients with oral squamous cell carcinoma (SCC) and was carried out using antibodies for MAGE-A4 and MAGE-C1. Fisher's exact test and log-rank test were used to evaluate the results. Results The expression of the MAGE-A4 and MAGE-C1 were 56.5% and 47.8% without statistical difference in studied variables and survival. Conclusion The expression of at least 1 CTA was present in 78.3% of the patients, however, without correlation with clinicopathologic variables and survival. (c) 2011 Wiley Periodicals, Inc. Head Neck, 2012