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PubMed2003

Radial patterning of Arabidopsis shoots by class III HD-ZIP and KANADI genes.

BACKGROUND: Shoots of all land plants have a radial pattern that can be considered to have an adaxial (central)-abaxial (peripheral) polarity. In Arabidopsis, gain-of-function alleles of PHAVOLUTA and PHABULOSA, members of the class III HD-ZIP gene family, result in adaxialization of lateral organs. Conversely, loss-of-function alleles of the KANADI genes cause an adaxialization of lateral organs. Thus, the class III HD-ZIP and KANADI genes comprise a genetic system that patterns abaxial-adaxial polarity in lateral organs produced from the apical meristem. RESULTS: We show that gain-of-function alleles of REVOLUTA, another member of the class III HD-ZIP gene family, are characterized by adaxialized lateral organs and alterations in the radial patterning of vascular bundles in the stem. The gain-of-function phenotype can be obtained by changing only the REVOLUTA mRNA sequence and without changing the protein sequence; this finding indicates that this phenotype is likely mediated through an interference with microRNA binding. Loss of KANADI activity results in similar alterations in vascular patterning as compared to REVOLUTA gain-of-function alleles. Simultaneous loss-of-function of PHABULOSA, PHAVOLUTA, and REVOLUTA abaxializes cotyledons, abolishes the formation of the primary apical meristem, and in severe cases, eliminates bilateral symmetry; these phenotypes implicate these three genes in radial patterning of both embryonic and postembryonic growth. CONCLUSIONS: Based on complementary vascular and leaf phenotypes of class III HD-ZIP and KANADI mutants, we propose that a common genetic program dependent upon miRNAs governs adaxial-abaxial patterning of leaves and radial patterning of stems in the angiosperm shoot. This finding implies that a common patterning mechanism is shared between apical and vascular meristems.

Arabidopsis↗

PubMed2005

Biodistribution of 68Ga-labelled phosphodiester, phosphorothioate, and 2'-O-methyl phosphodiester oligonucleotides in normal rats.

Antisense oligonucleotides may hybridise with high selectivity to mRNA sequences allowing monitoring of gene expression or inhibition of the manifestation of altered genes inducing diseases. As part of the development of positron emission tomography methods, 17-mer antisense phosphodiester (PO), phosphorothioate (PS) and 2'-O-methyl phosphodiester (OMe) oligonucleotides specific for point mutationally activated human K-ras oncogene were labelled with 68Ga radionuclide via a chelator coupled to the probe. Hybridisation in solution and non-denaturing polyacrylamide gel electrophoresis (PAGE) with a subsequent exposure of the gels was performed to verify the hybridisation ability after labelling. The biodistribution was studied in male Sprague-Dawley rats by injecting 2MBq of 68Ga-oligonucleotides via the tail vein and measuring the organ radioactivity concentration after 20, 60 and 120 min or using whole-body autoradiography with 10 MBq 68Ga-oligonucleotide and 20 min incubation time. Control experiments were performed with 68GaCl3 and 68Ga-chelator complex. The results revealed that 68Ga-labelling did not change the hybridisation abilities of the oligonucleotides. The biodistribution pattern depended on the nature of the oligonucleotide backbone. Bone marrow, kidney, liver, spleen and urinary bladder were the five organs of highest uptake with each oligonucleotide. The PO accumulated highly in the liver, whereas high kidney uptake dominated the PS and OMe patterns. Intact PS and OMe were detected in plasma samples taken 20 and 60 min after injection. This study supplies a base for the further development of 68Ga-labelled oligonucleotides as pharmacokinetic tools and a potential future use for in vivo imaging of gene expression.

Animals↗

PubMed2004

Regulating eukaryotic gene expression with aptamers.

Aptamers are RNA or DNA oligonucleotides identified within a randomly synthesized library, through an in vitro selection procedure. The selected candidates display a pre-determined property of interest with respect to a given target. Successful selection has been carried out against targets ranging from small (amino acids, antibiotics) to macro-molecules (proteins, nucleic acids). They generally show an affinity in the nanomolar range and a high specificity of target recognition. Interestingly, aptamers selected against purified targets in the test tube retain their properties within cells. RNA aptamers can be generated in situ from an appropriate DNA construct or delivered as nuclease-resistant oligonucleotide analogues. For example, aptamers recognizing RNA structure through loop-loop interactions modulate the trans-activation of in vitro transcription mediated by the TAR RNA element of human immunodeficiency virus type 1. Consequently, they constitute both exquisite tools for functional genomics analysis and promising prototypes of therapeutic agents. Natural aptameric motifs have been identified within mRNA sequences, which upon binding to a metabolite control the expression of the encoded gene, which is generally involved in the biosynthesis of this particular metabolite.

Amino Acid Motifs↗

PubMed2005

Deletions in L-type calcium channel alpha1 subunit testicular transcripts correlate with testicular cadmium and apoptosis in infertile men with varicoceles.

OBJECTIVE: To identify and understand predictors of successful varicocelectomy. DESIGN: Examination of testicular L-type voltage-dependent calcium channel (L-VDCC) mRNAs and proteins in testis biopsies and comparison of presence and absence of various mRNAs with testicular cadmium levels, with apoptosis, and with sperm count change after varicocelectomy. SETTING: University clinical urology practice and research laboratories. PATIENT(S): Infertile men with varicocele (left varicocele only, n = 18; bilateral varicoceles, n = 26) and controls (men with obstructive azoospermia undergoing testicular sperm extraction before intracytoplasmic sperm injection; n = 7). INTERVENTION(S): Left testis biopsies by percutaneous needle aspiration biopsy. Varicocele repair by subinguinal approach. MAIN OUTCOME MEASURE(S): Calcium channel mRNA sequence by reverse transcription-polymerase chain reaction and amplicon analysis; calcium channel protein distribution by immunocytochemistry; cadmium levels by atomic absorption and apoptosis by deoxynucleotidyl transferase labeling; and sperm counts in the ejaculate before and after varicocelectomy. RESULT(S): Calcium channel mRNAs are polymorphic in human testis biopsies from different men. Proteins from sequence-deleted exons 7 and/or 8 localize to germ cell membranes. Expression of undeleted L-type calcium channel mRNAs correlates with normal testes cadmium and increased sperm count after varicocelectomy. Apoptosis is lower in such cases. CONCLUSION(S): Expression of normal testicular L-VDCC sequence in exons 7-8 predicts postvaricocelectomy sperm count increase. Deletions may alter calcium channel function and affect testicular cadmium and apoptosis.

Adult↗

PubMed2006

NOX1/NADPH oxidase negatively regulates nerve growth factor-induced neurite outgrowth.

Reactive oxygen species produced by NADPH oxidase are involved in the neuronal death associated with various neurodegenerative disorders. However, the role of NADPH oxidase in neuronal differentiation has not been well characterized. In nondifferentiated PC12 cells, the mRNA level of NOX1, a catalytic subunit of NADPH oxidase expressed in nonphagocytes, was approximately 10 times higher than that of the phagocyte type subunit, NOX2 (gp91(phox)), while the transcript of another isoform, NOX4, was not detected. Following nerve growth factor (NGF)-induced neurite outgrowth, the mRNA level of NOX1 and NOX2 was progressively increased and decreased, respectively. The NGF-induced increase in NOX1 mRNA was mediated by TrkA and accompanied by increased intracellular superoxide, which was suppressed by NADPH oxidase inhibitors. Unexpectedly, these inhibitors and superoxide scavengers significantly enhanced NGF-induced neurite outgrowth. Enhanced neurite outgrowth was similarly demonstrated in cells depleted with the NOX1 transcript by stable expression of ribozymes targeted for the NOX1 mRNA sequence. Furthermore, NGF-induced expression of betaIII-tubulin was significantly augmented in cells treated with NADPH oxidase inhibitors or stably expressing ribozymes. Phosphatidylinositol-3 (PI3) kinase inhibitors, without affecting NGF-induced NOX1 expression, augmented NGF-induced neurite outgrowth but not in clones expressing ribozymes. Taken together, increased superoxide production by up-regulation of NOX1 may negatively regulate neuronal differentiation by suppressing excessive neurite outgrowth.

Animals↗

PubMed2006

Identification of novel splice variants of Adhesion G protein-coupled receptors.

Alternative splicing is an important mechanism to generate proteome diversity in higher eukaryotic organisms. We searched for splice variants of the human Adhesion family of G protein-coupled receptors (GPCRs) using mRNA sequences and expressed sequence tags. The results presented here describe 53 human splice variants among the 33 Adhesion GPCRs. Many of these variants appear to be coding for "functional" proteins (29) while the others are seemingly "non-functional" (24). Novel functional splice variants were found for: CD97, CELR3, EMR2, EMR3, GPR56, GPR110, GPR112-GPR114, GPR116, GPR123-GPR126, GPR133, HE6, and LEC1-LEC3. Splice variants for GPR116, GPR125, GPR126, and HE6 were found conserved in other species. Several of the functional splice variants lack one or more of the functional domains that are found in the N-termini of these receptors. These functional domains are likely to affect ligand binding or interaction with other proteins and these novel splice variants may have important roles for the specificity of interactions between these receptors and extracellular molecules. Another type of splice variants found here lacks a GPCR proteolytic site (GPS). The GPS domain has been shown to be essential for the proteolytic cleavage of the receptors N-termini and for cellular surface expression. We suggest that these alternative splice variants may be crucial for the function of the receptors while the seemingly non-functional splice variants may be a part of a regulative mechanism.

Alternative Splicing↗

PubMed2006

Immune cell recruitment following acoustic trauma.

Acoustic trauma induces cochlear inflammation. We hypothesized that chemokines are involved in the recruitment of leukocytes as part of a wound healing response. The cochleas of NIH-Swiss mice, exposed to octave-band noise (8-16 kHz, at 118 dB) for 2h, were examined after the termination of exposure. Leukocytes were identified immunohistochemically with antibodies to CD45 and F4/80. Gene array analysis followed by RT-PCR was performed on cochlear tissue to identify up-regulation of chemokine and adhesion molecule mRNA. The expression of the adhesion molecule ICAM-1 was also investigated immunohistochemically. Few CD45- or F4/80-positive leukocytes were observed in the non-exposed cochlea. Following acoustic trauma however, the number of CD45-positive cells was dramatically increased especially after 2 and 4 days, after which time the numbers decreased. F4/80-positive cells also increased in number over the course of a week. Gene array analysis indicated increased expression of monocyte chemoattractant protein 5 (MCP-5), monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein-1beta (MIP-1beta) and ICAM-1. RT-PCR, performed using primers for the individual mRNA sequences, confirmed the increased expression of MCP-1, MCP-5, MIP-1beta, and ICAM-1 relative to non-exposed mice. In the normal cochlea, ICAM-1 immunohistochemical expression was observed in venules, spiral ligament fibrocytes and in endosteal cells of the scala tympani. Expression increased to include more of the spiral ligament and endosteal cells after acoustic trauma. A cochlear inflammatory response is initiated in response to acoustic trauma and involves the recruitment of circulating leukocytes to the inner ear.

Animals↗

PubMed2004

Direct quantification of mRNA expression levels using single molecule detection.

Determination of the gene expression by direct quantification of mRNA is becoming increasingly important in basic, pharmaceutical and clinical research. We present a novel approach for gene quantification based on direct hybridization of gene-specific probes to target mRNA sequences in solution at temperatures ensuring absolute specificity of the probe-target complex. No enzymatic steps like reverse transcription or amplification by PCR are involved within the quantification process. In order to increase specificity as well as sensitivity, two probes emitting fluorescence light in different colors are used for our homogeneous assay using fluorescence cross-correlation. We relate to the single molecule sensitivity of excitation and detection in confocal cavities avoiding the amplification of the detected signal. The analysis of the expression level of high, medium and low abundant genes is described in two different cell lines, whereby the genes are quantified in absolute numbers.

Calcium-Binding Proteins↗

PubMed2004

Identification of putative rat ribonuclease III by differential display: a novel rat mRNA expressed in a circadian manner in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) of the hypothalamus constitutes the principal site responsible for the generation and entrainment of circadian rhythms in mammals. The mechanisms of the circadian clock involve periodic gene expression. Here we report the use of differential display reverse transcriptase polymerase chain reaction to identify a novel rat mRNA sequence which is highly homologous to human ribonuclease III. Analysis of its expression in the rat brain by in situ hybridization histochemistry showed this transcript to be expressed at differing intensities at various sites. Temporal variation in expression was observed in the SCN, with a peak at circadian time (CT) 2 and a nadir at CT14. No significant changes in its expression were detected across the cycle within the supraoptic nucleus, cingulate cortex or caudate putamen.

Animals↗

PubMed2005

Elongation factors on the ribosome.

The ribosome is a complex macromolecular assembly capable of translating mRNA sequence into amino acid sequence. The adaptor molecule of translation is tRNA, but the delivery of aminoacyl-tRNAs--the primary substrate of the ribosome--relies on the formation of a ternary complex with elongation factor Tu (EF-Tu) and GTP. Likewise, elongation factor G (EF-G) is required to reset the elongation cycle through the translocation of tRNAs. Recent structures and biochemical data on ribosomes in complex with the ternary complex or EF-G have shed light on the mode of action of the elongation factors, and how this interplays with the state of tRNAs and the ribosome. A model emerges of the specific routes of conformational changes mediated by tRNA and the ribosome that trigger the GTPase activity of the elongation factors on the ribosome.

Binding Sites↗

PubMed2004

Therapeutic siRNAs.

The ability of small-interfering RNAs (siRNAs) to silence gene expression in somatic mammalian cells has provided researchers with a novel tool to block the expression of disease-causing genes, provided that their mRNA sequences are known. siRNAs can be delivered to cells either exogenously as synthetic agents or endogenously as gene-encoding siRNAs. Recent studies demonstrate the general application of siRNAs to silence gene expression in a range of cell types and in whole mammals. Beyond their value for dissecting gene functions and target validation, siRNAs also hold great potential as gene-specific therapeutic agents.

Animals↗

PubMed2004

The complete genomic organization of the human MUC6 and MUC2 mucin genes.

The complete genomic organization of the two mucin genes MUC2 and MUC6 was obtained by comparison of new and published mRNA sequences with newly available human genomic sequence. The two genes are located 38.5 kb apart in a head-to-head orientation within a gene complex on chromosome 11p15.5. The N-terminal organization of MUC6 is highly similar to that of MUC2, containing the D1, D2, D', and D3 Von Willebrand factor domains followed by the large tandem repeat domains located in exons 31 and 30, respectively. MUC6 has a much smaller C-terminal domain (101 amino acids) encoded by 2 exons containing only the CK domain, compared with MUC2, which has a C-terminal domain of 859 amino acids containing the D4, C, D, and CK domains, encoded by 19 exons. The gene structures agreed partially but not completely with predictions from gene prediction programs.

Chromosomes, Human, Pair 11↗

PubMed2002

Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue.

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.

Adenocarcinoma↗

PubMed2004

Calpain mediates calcium-induced activation of the erk1,2 MAPK pathway and cytoskeletal phosphorylation in neurons: relevance to Alzheimer's disease.

Aberrant phosphorylation of the neuronal cytoskeleton is an early pathological event in Alzheimer's disease (AD), but the underlying mechanisms are unclear. Here, we demonstrate in the brains of AD patients that neurofilament hyperphosphorylation in neocortical pyramidal neurons is accompanied by activation of both Erk1,2 and calpain. Using immunochemistry, Western blot analysis, and kinase activity measurements, we show in primary hippocampal and cerebellar granule (CG) neurons that calcium influx activates calpain and Erk1,2 and increases neurofilament phosphorylation on carboxy terminal polypeptide sites known to be modulated by Erk1,2 and to be altered in AD. Blocking Erk1,2 activity either with antisense oligonucleotides to Erk1,2 mRNA sequences or by specifically inhibiting its upstream activating kinase MEK1,2 markedly reduced neurofilament phosphorylation. Calpeptin, a cell-permeable calpain inhibitor, blocked both Erk1,2 activation and neurofilament hyperphosphorylation at concentrations that inhibit calpain-mediated cleavage of brain spectrin. By contrast, inhibiting Erk1,2 with U-0126, a specific inhibitor of Mek1,2, had no appreciable effect on ionomycin-induced calpain activation. These findings demonstrate that, under conditions of calcium injury in neurons, calpains are upstream activators of Erk1,2 signaling and are likely to mediate in part the hyperphosphorylation of neurofilaments and tau seen at early stages of AD as well as the neuron survival-related functions of the MAP kinase pathway.

Adult↗

PubMed1992

Leukocyte adhesion deficiency: identification of novel mutations in two Japanese patients with a severe form.

Leukocyte adhesion deficiency is a disorder with mutations of the gene for the beta subunit, a component common to three adhesion molecules; LFA-1, Mac-1 and p150,95. The molecular basis of the disorder was studied in two patients with its severe form. In the first patient, the mutant gene expressed an aberrant mRNA, 1.2 kb longer than usual, resulting from a G to A substitution at the splice donor site of a 1.2 kb intron. Several aberrantly spliced messages, arising from splicing at cryptic donor sites, were also identified. The beta subunit proteins deduced from the mRNA sequences lacked half the carboxyl terminal portion. In the second patient, the mutation was a G to A transition at nucleotide 454, which resulted in an Asp128 to Asn substitution of the beta subunit. The 128th Asp residue is located in a region crucial for the association with alpha subunits and strictly conserved among the integrin beta subunits so far analyzed.

Adult↗

PubMed2003

Alterations in cellular IRP-dependent iron regulation by in vitro manganese exposure in undifferentiated PC12 cells.

Manganese (Mn) may interfere with iron regulation by altering the binding of iron regulatory proteins (IRPs) to their response elements found on the mRNA encoding proteins critical to iron homeostasis. To explore this, the effects of 24-h in vitro manganese exposure (1, 10, 50, and 200 microM Mn) on: (i) total intracellular and labile iron concentrations; (ii) the cellular abundance of transferrin receptor (TfR), H- and L-ferritin, and mitochondrial aconitase proteins; and (iii) IRP binding to a [32P](-) labeled mRNA sequence of L-ferritin were evaluated in undifferentiated PC12 cells. In vitro manganese exposure altered the cellular abundance of TfR, H-/L-ferritin, and m-aconitase, resulting in an increase in labile iron. This latter effect led to a decrease in IRP binding activity at the lower (10 and 50 microM) manganese exposures. In contrast, 200 microM manganese exposure increased IRP binding, in spite of the significant increase in labile iron. These data indicate that at lower exposures, manganese directly interfered with IRP-dependent translational events, producing an increase in labile iron, which in turn signaled a decrease in IRP binding at 24 h. At higher exposures, the intracellular burden of manganese resulted in overt cytotoxicity and appeared to compromise the normal compensatory response to increased labile iron, producing increased IRP binding. We conclude that low to moderate manganese exposure interferes with cellular iron regulation, and thus may serve as a contributory mechanism underlying manganese neurotoxicity.

Animals↗

PubMed1999

Correlation of Fos expression and circling asymmetry during gerbil vestibular compensation.

Vestibular compensation is a central nervous system process resulting in recovery of functional movement and control following a unilateral vestibular lesion. Small pressure injections of phosphorothioate 20mer oligonucleotides were used to probe the role of the Fos transcription protein during vestibular compensation in the gerbil brainstem. During isoflurane gas anesthesia, antisense probes against the c-fos mRNA sequence were injected into the medial vestibular and prepositus nuclei unilaterally prior to a unilateral surgical labyrinthectomy. Anionic dyes, which did not interact with the oligonucleotides, were used to mark the injection site and help determine the extent of diffusion. The antiFos oligonucleotide injections reduced Fos expression at the injection site in neurons which normally express Fos after the lesion, and also affected circling behavior induced by hemilabyrinthectomy. With both ipsilateral and contralateral medial vestibular and prepositus nuclei injections, less ipsilateral and more contralateral circling was noted in animals injected with antiFos injections as compared to non-injected controls. The degree of change in these behaviors was dependent upon the side of the injection. Histologically, antiFos injections reduced the number of Fos immunolabeled neurons around the injection site, and increased Fos expression contralaterally. The correlation of the number of neurons with Fos expression to turning behavior was stronger for contralateral versus ipsilateral turns, and for neurons in the caudal and ipsilateral sub-regions of the medial vestibular and prepositus nuclei. The results are discussed in terms of neuronal firing activity versus translational activity based on the asymmetrical expression of the Fos inducible transcription factor in the medial vestibular and prepositus nuclei. Although ubiquitous in the brain, transcription factors like Fos can serve localized and specific roles in sensory-specific adaptive stimuli. Antisense injections can be an effective procedure for localized intervention into complex physiological functions, e.g. vestibular compensation.

Adaptation, Physiological↗

PubMed2002

Estrogen receptor beta. Potential functional significance of a variety of mRNA isoforms.

Recent cloning of estrogen receptor beta (ERbeta) was followed by the discovery of a variety of its isoforms. This review describes the complexity of ERbeta mRNAs in various species for which most data have been gathered so far. The most surprising finding is the great variation in isoform structure among various mammalian species. This may reflect either the fact that only a very limited number of isoforms have been described so far or between-species specificity, especially as common elements in closely related species could still be noted. Isoform variations, as detected mainly at the mRNA sequence level, should result in profound functional differences at the level of proteins as already shown in selected cases. Thus, it is proposed that the diversity of ERbeta isoforms implies a functional role of this phenomenon in cellular physiology and pathology of estrogen response.

Animals↗