New computational biology tools for the systematic analysis of the structure and expression of human genes

From the late 1980s, the automation of sequencing techniques and the computer spread gave rise to a flourishing number of new molecular structures and sequences and to proliferation of new databases in which to store them. Here are presented three computational approaches able to analyse the massive amount of publicly avalilable data in order to answer to important biological questions. The first strategy studies the incorrect assignment of the first AUG codon in a messenger RNA (mRNA), due to the incomplete determination of its 5' end sequence. An extension of the mRNA 5' coding region was identified in 477 in human loci, out of all human known mRNAs analysed, using an automated expressed sequence tag (EST)-based approach. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 and the consequences for the functional studies are discussed. The second approach analyses the codon bias, the phenomenon in which distinct synonymous codons are used with different frequencies, and, following integration with a gene expression profile, estimates the total number of codons present across all the expressed mRNAs (named here "codonome value") in a given biological condition. Systematic analyses across different pathological and normal human tissues and multiple species shows a surprisingly tight correlation between the codon bias and the codonome bias. The third approach is useful to studies the expression of human autism spectrum disorder (ASD) implicated genes. ASD implicated genes sharing microRNA response elements (MREs) for the same microRNA are co-expressed in brain samples from healthy and ASD affected individuals. The different expression of a recently identified long non coding RNA which have four MREs for the same microRNA could disrupt the equilibrium in this network, but further analyses and experiments are needed.

MicroRNA-381 Represses ID1 and is Deregulated in Lung Adenocarcinoma

MicroRNAs are small, noncoding RNAs that suppress gene expression by binding to the 3' untranslated region (UTR) and thereby repress translation or decrease messenger RNA stability. Inhibitor of differentiation 1 (ID1) is a putative stem-cell gene involved in invasion and angiogenesis. We previously showed that ID1 is regulated by Src kinases, overexpressed in human lung adenocarcinoma, and targeted by Src-dependent microRNAs. The current study focused on the association between miR-381 and ID1 in lung adenocarcinoma.

Hint2, a mitochondrial apoptotic sensitizer down-regulated in hepatocellular carcinoma

BACKGROUND ; AIMS: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2. METHODS: Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays. RESULTS: Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival. CONCLUSION: Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.

Plakoglobin deficiency protects keratinocytes from apoptosis

The armadillo family protein plakoglobin (Pg) is a well-characterized component of anchoring junctions, where it functions to mediate cell-cell adhesion and maintain epithelial tissue integrity. Although its closest homolog beta-catenin acts in the Wnt signaling pathway to dictate cell fate and promote proliferation and survival, the role of Pg in these processes is not well understood. Here, we investigate how Pg affects the survival of mouse keratinocytes by challenging both Pg-null cells and their heterozygote counterparts with apoptotic stimuli. Our results indicate that Pg deletion protects keratinocytes from apoptosis, with null cells exhibiting delayed mitochondrial cytochrome c release and activation of caspase-3. Pg-null keratinocytes also exhibit increased messenger RNA and protein levels of the anti-apoptotic molecule Bcl-X(L) compared to heterozygote controls. Importantly, reintroduction of Pg into the null cells shifts their phenotype towards that of the Pg+/- keratinocytes, providing further evidence that Pg plays a direct role in regulating cell survival. Taken together, our results suggest that in addition to its adhesive role in epithelia, Pg may also function in contrast to the pro-survival tendencies of beta-catenin, to potentiate death in cells damaged by apoptotic stimuli, perhaps limiting the potential for the propagation of mutations and cellular transformation.Journal of Investigative Dermatology advance online publication, 16 November 2006; doi:10.1038/sj.jid.5700615.

Galectin-3 modulates T cell activity and is reduced in the inflamed intestinal epithelium in IBD

BACKGROUND: Galectins are involved at different stages in inflammation. Galectin-3, although mostly described as proinflammatory, can also act as an immunomodulator by inducing apoptosis in T cells. The present study aims to determine galectin-3 expression in the normal and inflamed intestinal mucosa and to define its role in T cell activity. MATERIALS AND METHODS: Galectin-3 was detected by quantitative polymerase chain reaction with total RNA from endoscopic biopsies and by immunohistochemistry. Biopsies and peripheral blood mononuclear cells (PBMC) were stimulated in vitro and were used to assess the functional consequences of inhibition or exogenous addition of galectin-3. RESULTS: Galectin-3 is expressed at comparable levels in controls and inflammatory bowel disease (IBD) patients in remission. In the normal mucosa, galectin-3 protein was mainly observed in differentiated enterocytes, preferentially at the basolateral side. However, galectin-3 was significantly downregulated in inflamed biopsies from IBD patients. Ex vivo stimulation of uninflamed biopsies with tumor necrosis factor led to similar galectin-3 messenger RNA downregulation as in vivo. When peripheral blood mononuclear cells (PBMC) were analyzed, galectin-3 was mainly produced by monocytes. Upon mitogen stimulation, we observed increased proliferation and decreased activation-induced cell death of peripheral blood T cells in the presence of galectin-3-specific small interfering RNA. In contrast, exogenous addition of recombinant galectin-3 led to reduced proliferation of mitogen-stimulated peripheral blood T cells. CONCLUSIONS: Our results suggest that downregulation of epithelial galectin-3 in the inflamed mucosa reflects a normal immunological consequence, whereas under noninflammatory conditions, its constitutive expression may help to prevent inappropriate immune responses against commensal bacteria or food compounds. Therefore, galectin-3 may prove valuable for manipulating disease activity.

Differential regulation of metzincins in experimental chronic renal allograft rejection: potential markers and novel therapeutic targets

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-beta1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin alpha/beta had decreased. Accordingly, MMP-9 protein levels and meprin alpha/beta synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.

Malignant melanoma and bone resorption

The cellular and humoral mechanisms accounting for osteolysis in skeletal metastases of malignant melanoma are uncertain. Osteoclasts, the specialised multinucleated cells that carry out bone resorption, are derived from monocyte/macrophage precursors. We isolated tumour-associated macrophages (TAMs) from metastatic (lymph node/skin) melanomas and cultured them in the presence and absence of osteoclastogenic cytokines and growth factors. The effect of tumour-derived fibroblasts and melanoma cells on osteoclast formation and resorption was also analysed. Melanoma TAMs (CD14+/CD51-) differentiated into osteoclasts (CD14-/CD51+) in the presence of receptor activator for nuclear factor kappaB ligand (RANKL) and macrophage-colony stimulating factor. Tumour-associated macrophage-osteoclast differentiation also occurred via a RANKL-independent pathway when TAMs were cultured with tumour necrosis factor-alpha and interleukin (IL)-1alpha. RT-PCR showed that fibroblasts isolated from metastatic melanomas expressed RANKL messenger RNA and the conditioned medium of cultured melanoma fibroblasts was found to be capable of inducing osteoclast formation in the absence of RANKL; this effect was inhibited by the addition of osteoprotegerin (OPG). We also found that cultured human SK-Mel-29 melanoma cells produce a soluble factor that induces osteoclast differentiation; this effect was not inhibited by OPG. Our findings indicate that TAMs in metastatic melanomas can differentiate into osteoclasts and that melanoma fibroblasts and melanoma tumour cells can induce osteoclast formation by RANKL-dependent and RANKL-independent mechanisms, respectively.

Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors on human platelets

Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.

CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients >or=60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients.

Detection of perforin and granzyme B mRNA expressing cells in lichen sclerosus

Granzyme B and perforin messenger RNA (mRNA) expression has been shown to be a specific in vivo activation marker for cytotoxic cells. The aim of this study was to assess the contribution of cell-mediated cytotoxicity in the pathogenesis of lichen sclerosus. In situ hybridization and immunohistochemistry were performed on serial tissue sections of lesional skin biopsies and normal skin as control. Immunohistochemical staining showed that the cellular infiltrate of diseased skin consisted predominantly of T cells (CD3+) and some B cells (CD20+). Among T cells CD4+ and CD8+ cells were found in about equal numbers. In normal skin samples perforin and granzyme B mRNA expressing cells were only rarely found. In contrast, in biopsies from diseased skin a high percentage of infiltrating cells expressed mRNA for perforin and granzyme B. The perforin and granzyme B expressing cells were found in the dermal infiltrate and intraepidermally in close proximity to keratinocytes suggesting in situ activation of these cells. These findings provide evidence that cell-mediated cytotoxicity plays a significant role in tissue destruction in lichen sclerosus.

Identification of markers to characterize and sort human articular chondrocytes with enhanced in vitro chondrogenic capacity

OBJECTIVE: To identify markers associated with the chondrogenic capacity of expanded human articular chondrocytes and to use these markers for sorting of more highly chondrogenic subpopulations. METHODS: The chondrogenic capacity of chondrocyte populations derived from different donors (n = 21) or different clonal strains from the same cartilage biopsy specimen (n = 21) was defined based on the glycosaminoglycan (GAG) content of tissues generated using a pellet culture model. Selected cell populations were analyzed by microarray and flow cytometry. In some experiments, cells were sorted using antibodies against molecules found to be associated with differential chondrogenic capacity and again assessed in pellet cultures. RESULTS: Significance Analysis of Microarrays indicated that chondrocytes with low chondrogenic capacity expressed higher levels of insulin-like growth factor 1 and of catabolic genes (e.g., matrix metalloproteinase 2, aggrecanase 2), while chondrocytes with high chondrogenic capacity expressed higher levels of genes involved in cell-cell or cell-matrix interactions (e.g., CD49c, CD49f). Flow cytometry analysis showed that CD44, CD151, and CD49c were expressed at significantly higher levels in chondrocytes with higher chondrogenic capacity. Flow cytometry analysis of clonal chondrocyte strains indicated that CD44 and CD151 could also identify more chondrogenic clones. Chondrocytes sorted for brighter CD49c or CD44 signal expression produced tissues with higher levels of GAG per DNA (up to 1.4-fold) and type II collagen messenger RNA (up to 3.4-fold) than did unsorted cells. CONCLUSION: We identified markers that allow characterization of the capacity of monolayer-expanded chondrocytes to form in vitro cartilaginous tissue and enable enrichment for subpopulations with higher chondrogenic capacity. These markers might be used as a means to predict and possibly improve the outcome of cell-based cartilage repair techniques.

Neurokinin-2 receptor levels correlate with intensity, frequency, and duration of pain in chronic pancreatitis

OBJECTIVE: Generation and maintenance of pain in chronic pancreatitis (CP) have been shown to be partially attributable to neuroimmune interactions, which involve neuropeptides such as substance P (SP). So far, expression of SP receptors NK-2R, NK-3R, the SP-encoding gene preprotachykinin A (PPT-A), and the SP degradation enzyme neutral endopeptidase (NEP) and their relation to pain in CP have not been determined. METHODS: Tissue samples from patients with CP (n = 25) and from healthy donors (n = 20) were analyzed for PPT-A, NK-2R, NK-3R, and NEP expression using quantitative RT-PCR. NEP protein levels were examined by immunoblot analysis and its localization was determined using immunohistochemistry. A scoring system was used to grade the extent of fibrosis on hematoxylin and eosin- and Masson-Trichrome-stained sections. Messenger RNA levels and the extent of pain were analyzed for correlations. RESULTS: In CP tissues, NK-2R and PPT-A expression was increased, whereas NK-3R and NEP mRNA levels were comparable with normal pancreas. Overexpression of NK-2R was related to the intensity, frequency, and duration of pain in CP patients. NK-1R and NEP expression was significantly related to the extent of fibrosis. CONCLUSIONS: Expression of NK-2R and PPT-A is increased in CP and is associated with pain. Failure to up-regulate NEP may contribute to the disruption of the neuropeptides loop balance in CP and thus may exacerbate the severe pain syndrome.

Comparative quantitative assessment of GnRH- and LH-receptor mRNA expression in the urinary tract of sexually intact and spayed female dogs

Ovariectomy interrupts the regulatory loop in the hypothalamus-pituitary-gonad axis, leading to a several-fold increase in gonadotropin levels. This rise in hormonal secretion may play a causal role in ovariectomy-related urinary incontinence. The purpose of this study was to examine the effect of ovariectomy in bitches on the expression of GnRH- and LH-receptors in the lower urinary tract, and assess the relationship between receptor expression and plasma gonadotropin concentrations. Plasma gonadotropins were measured in 37 client-owned bitches. Biopsies were harvested from the mid-ventral bladder wall in all dogs, and from nine further locations within the lower urinary tract in 17 of the 37 animals. Messenger RNA of the LH and GnRH receptors was quantified using RT-PCR with the TaqMan Universal PCR Master Mix. Gonadotropins were measured with a canine-specific FSH-immunoradiometric assay and LH-radioimmunoassay. The hierarchical mixed ANOVA model using MINITAB, Mann-Whitney U-test, unpaired means comparison and linear regressions using StatView were applied for statistical analyses. Messenger RNA for both receptors was detected in all biopsy samples. Age was negatively correlated to mRNA expression of the LH and the GnRH receptors. A relationship between the mRNA values and the plasma gonadotropin concentrations was not established. Evaluation of results within each of the biopsy locations revealed greater LH-receptor expression in the proximal second quarter of the urethra in spayed bitches than in intact bitches (P=0.0481). Increased mRNA expression of LH receptors in this location could possibly play a role in the decrease in closing pressure of the urethra following ovariectomy.

Spinal muscular atrophy: SMN2 pre-mRNA splicing corrected by a U7 snRNA derivative carrying a splicing enhancer sequence

Spinal muscular atrophy (SMA) is a lethal hereditary disease caused by homozygous deletion/inactivation of the survival of motoneuron 1 (SMN1) gene. The nearby SMN2 gene, despite its identical coding capacity, is only an incomplete substitute, because a single nucleotide difference impairs the inclusion of its seventh exon in the messenger RNA (mRNA). This splicing defect can be corrected (transiently) by specially designed oligonucleotides. Here we have developed a more permanent correction strategy based on bifunctional U7 small nuclear RNAs (snRNAs). These carry both an antisense sequence that allows specific binding to exon 7 and a splicing enhancer sequence that will improve the recognition of the targeted exon. When expression cassettes for these RNAs are stably introduced into cells, the U7 snRNAs become incorporated into small nuclear ribonucleoprotein (snRNP) particles that will induce a durable splicing correction. We have optimized this strategy to the point that virtually all SMN2 pre-mRNA becomes correctly spliced. In fibroblasts from an SMA patient, this approach induces a prolonged restoration of SMN protein and ensures its correct localization to discrete nuclear foci (gems).

Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.