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At least 1,405 records · Page 78Linked to original sources
PubMed2005

Characterization of the nucleic acid-binding activity of the avian reovirus non-structural protein sigma NS.

The avian reovirus non-structural protein sigma NS has previously been shown to bind single-stranded (ss) RNA in vitro in a sequence-independent manner. The results of the present study further reveal that sigma NS binds poly(A), poly(U) and ssDNA, but not poly(C), poly(G) or duplex nucleic acids, suggesting that sigma NS has some nucleotide-sequence specificity for ssRNA binding. The current findings also show that sigma NS is present in large ribonucleoprotein complexes in the cytoplasm of avian reovirus-infected cells, indicating that it exists in intimate association with ssRNAs in vivo. Removal of RNA from the complexes generates a sigma NS protein form that sediments between 4.5 and 7 S, suggesting that RNA-free sigma NS associates into small oligomers. Expression and purification of recombinant sigma NS in insect cells allowed us to generate specific antibodies and to perform a variety of assays. The results of these assays revealed that: (i) RNA-free sigma NS exists as homodimers and homotrimers; (ii) the minimum RNA size for sigma NS binding is between 10 and 20 nt; (iii) sigma NS does not have a preference for viral mRNA sequences; and (iv) its RNA-binding activity is conformation-dependent. Baculovirus expression of point and deletion sigma NS mutants in insect cells showed that the five conserved basic amino acids that are important for RNA binding and ribonucleoprotein-complex formation are dispersed throughout the entire sigma NS sequence, suggesting that this protein binds ssRNA through conformational domains. Finally, the properties of the avian reovirus protein sigma NS are compared with those of its mammalian reovirus counterpart.

Amino Acid Sequence↗
PubMed1985

Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli.

Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.

Adenosine Deaminase↗
PubMed1988

Absence of a gamma-melanocyte-stimulating hormone sequence in proopiomelanocortin mRNA of chum salmon Oncorhynchus keta.

1. Complete nucleotide sequence of one of the salmon proopiomelanocortin mRNAs (POMC mRNAs) was determined. 2. The region corresponding to gamma-melanocyte-stimulating hormone (gamma-MSH) was lacking in salmon POMC mRNA, although overall organization of the multi-hormone structure was exactly the same as that of mammalian POMC mRNAs. 3. The possible evolutional history of POMC mRNA in mammalian species may be revealed from the finding of this characteristic that salmon POMC mRNA lacks the region corresponding to gamma-MSH.

Amino Acid Sequence↗
PubMed1992

Simultaneous in situ hybridisation of native mRNA and immunoglobulin detection by conventional immunofluorescence in paraffin wax embedded sections.

AIMS: The development of a technique for simultaneous in situ hybridisation for native mRNA and conventional immunofluorescence for cytoplasmic antigens in routine pathology specimens. METHODS: Cocktails of synthetic deoxyoligonucleotides coding for immunoglobulin J chain and kappa light chain were 3' end labelled enzymatically with digoxigenin using terminal deoxynucleotidyl transferase. Native mRNA sequences were "unmasked" using proteolytic digestion with proteinase K and hybrid detection was achieved with an alkaline phosphatase labelled anti-digoxigenin antibody. Alkaline phosphatase was visualised with Fast red/naphthol AS-MX phosphate. Fluorescein isothiocyanate (FITC) conjugated anti-isotype antibodies were used simultaneously at the detection stage to identify the isotype production by individual plasma cells in endoscopic duodenal biopsy specimens. RESULTS: The IgA plasma cells of the lamina propria were identified by immunofluorescence and hybrids were detected in the anticipated plasma cell population by Fast red visualisation. The reaction product was visible in bright field or ultraviolet illumination which allowed FITC and Fast red labels to be visualised together under ultraviolet light at 490 nm. Dual labelled cells were clearly visible. Morphology was well preserved throughout. CONCLUSIONS: This technique permits the demonstration of specific mRNA species in cells expressing immunoglobulin. It combines all the advantages of non-radioactive synthetic oligonucleotide probes and conventional immunofluorescence techniques in routine formol-saline fixed and paraffin wax embedded sections with good retention of morphology.

Antigens↗
PubMed1985

The 1A protein gene of human respiratory syncytial virus: nucleotide sequence of the mRNA and a related polycistronic transcript.

The 1A mRNA is the smallest mRNA of human respiratory syncytial (RS) virus and encodes a single protein of approximate molecular weight 9500 as estimated previously by gel electrophoresis. The nucleotide sequence of the 1A mRNA, determined from several full-length cDNA clones, is reported. The 1A mRNA consists of 405 nucleotides, exclusive of poly(A), with relatively long nontranslated regions at the 5' and 3' ends (84 and 126 nucleotides, respectively). The sequences at the 5' and 3' termini of the 1A mRNA conform to the previously described conserved consensus sequences for RS virus mRNAs. The major open reading frame of the 1A mRNA codes for a hydrophobic polypeptide of 64 amino acids with a calculated molecular weight of 7536. The 5' terminus of the 1A mRNA was mapped and sequenced by primer extension under conditions for sequencing by partial chain termination. These experiments also identified a population of polycistronic RNA having the general structure: 5' M protein mRNA-1A mRNA 3'. This polytranscript was sequenced in order to determine the intergenic sequence. In the polytranscript, the nucleotide sequence of the M gene is followed by, in mRNA sense, six A residues and the intergenic sequence 5' ... UAUACACNN (N represents unidentified nucleotide).

Amino Acid Sequence↗
PubMed2002

Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract.

The coding sequence within several mitochondrial mRNAs of the trypanosomatid protozoa is created through editing by the precise insertion and deletion of U nucleotides. The biochemical characterisation of the editing reaction in the Leishmania genus of the trypanosomatids has been hindered by the lack of a direct in vitro assay. We describe here the first direct assay for the detection of guide RNA-directed editing mediated by a mitochondrial extract prepared from two independent isolates of Leishmania tarentolae. The assay enabled the editing activity within a L. tarentolae mitochondrial extract to be significantly enriched and will facilitate the characterisation of the editing reaction. The results suggest that the difficulty in establishing an assay for the L. tarentolae reaction was not simply a result of the catalytic machinery being limiting but rather reflected the presence of constraints on both the guide RNA and mRNA sequences.

Animals↗
PubMed2003

SPEER--a new family of testis-specific genes from the mouse.

Differential cloning revealed a partial mRNA sequence expressed in the mouse testis, which on further molecular characterization proved to be a member of a new family of 14 transcribed genes. Six of the genes appear to be expressed pseudogenes. The remainder indicate an open reading frame of approximately 200-220 amino acids encoding proteins with a very high proportion of alpha helical secondary structure, comprising approximately 15% glutamate residues. Because of this property, the family has been named SPErm-associated glutamate (E)-Rich protein (SPEER). Three members were chosen for more detailed characterization: SPEER-1 (pseudogene), SPEER-2, and SPEER-4D. All three are expressed tissue specifically in the testis of mice, with only very weak expression evident in the rat testis but in no other species tested. Using reverse transcription-polymerase chain reaction (RT-PCR), all three transcripts can be detected also in the epididymis, presumably due to the presence of spermatozoa. All three transcripts are expressed to high levels in haploid germ cells at the spermatocyte-spermatid transition. SPEER-1 mRNA is present in the cytoplasm as a sense transcript, SPEER-2 appears to be made mostly as an antisense transcript, whereas SPEER-4D appears to be localized within a subcellular compartment as a conventional sense transcript. Codon usage analysis suggests that all but the pseudogenes can be expressed as protein, confirmed for SPEER-2 and SPEER-4D by in vitro transcription/translation. An antibody raised against a peptide region of SPEER-4D, which probably cross-reacts with other SPEER members, immunohistochemically stains the nuclei of early round spermatids. While there are no true homologies to other proteins in the genome databases, some motifs are present that suggest a relationship to nuclear matrix proteins, implying that the SPEER family is a new group of haploid sperm-specific nuclear factors.

Amino Acid Sequence↗
PubMed2002

Identity elements used in export of mRNAs.

Different classes of RNA are exported from the nucleus by distinct factors. We demonstrate that U1 snRNA is exported like an mRNA on insertion of a pre-mRNA intron or either sense or antisense mRNA exon sequences. mRNA-specific factors are recruited onto the spliced or elongated U1 RNA whereas U snRNA-specific factors are not, suggesting that an unstructured region of sufficient length in an RNA acts as a dominant determinant of mRNA identity. After export, spliced U1 RNA undergoes cytoplasmic maturation but is not reimported into the nucleus. These data provide insight into mechanisms for discrimination of different classes of nuclear RNA and demonstrate that two RNAs of identical sequence can have distinct cytoplasmic fates depending on their mode of export.

Active Transport, Cell Nucleus↗
PubMed1998

Expression of functional estrogen receptors and galanin messenger ribonucleic acid in immortalized luteinizing hormone-releasing hormone neurons: estrogenic control of galanin gene expression.

The activity of estradiol on the LHRH neuronal network is crucial in the regulation of reproduction. In vivo, estradiol induces galanin (GAL) gene expression in LHRH neurons and GAL/LHRH colocalization is sexually dimorphic and neonatally determined by steroid exposure. The effects of estradiol on LHRH neurons, however, are considered to be indirect because estrogen receptors (ER) have not been detected in LHRH neurons in vivo. Using immortalized mouse LHRH neurons (GT1-7 cells), we demonstrated by RT-PCR and Southern blotting that GT1-7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indicated that GT1-7 ER is of the alpha-subtype (ER alpha). Additionally, estrogen receptors in GT1-7 cells were characterized by competitive radioligand receptor binding and IC50 values for 17beta-estradiol and ICI-182,780 were found to be 0.24 and 4.1 nM, respectively. The ability of endogenous GT1-7 cell ER to regulate transcription was determined in transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response elements (ERE) and a minimal herpes simplex virus thymidine kinase promoter. 17Beta-estradiol was found to enhance luciferase activity by 2.5-fold at physiological concentrations with an ED50 value of 47 pM. This induction was completely inhibited by ICI-182,780 which had an IC50 value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and droloxifene also fully blocked estrogen-mediated luciferase induction with IC50 values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In addition, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17beta-estradiol to modulate expression of the endogenous GAL gene in immortalized LHRH neurons was also determined. Quantitative RT-PCR demonstrated that physiological concentrations of estrogen increase GAL gene expression by 2-fold with an ED50 value of 23 pM. ICI-182,780, raloxifene, and droloxifene completely blocked this induction. In summary, our data demonstrate the presence of ER alpha and GAL mRNA in GT1-7 cells. The ER in GT1-7 cells is biologically active because 17beta-estradiol enhances both endogenous GAL gene expression and an ERE-driven reporter gene. These results suggest that estrogenic control of GAL gene expression in immortalized LHRH neurons may be transduced by ER. Thus, hypothalamic-derived LHRH neurons appear to have the capacity to be directly regulated by estrogen.

Animals↗
PubMed1983

The transposition unit of variant surface glycoprotein gene 118 of Trypanosoma brucei. Presence of repeated elements at its border and absence of promoter-associated sequences.

At the DNA level, antigenic variation in trypanosomes is brought about by the replacement of one variant surface glycoprotein (VSG) gene by another in an expression site with a strong promoter. In several cases studied, mobilization of a VSG gene for expression involves a duplication-transposition. We have determined the DNA sequence of most of the transposed segment of one such VSG, the VSG 118. At the 3' side, the transposed segment ends within the end of the gene; at the 5' side, the transposed segment is preceded by a putative VSG gene, extending our previous conclusion that VSG genes are tightly clustered. The total length of the transposed segment is about 3.5 X 10(3) base-pairs and 1.8 X 10(3) base-pairs of this codes for the VSG 118 messenger RNA. Near the 5' border of the transposed segment we find five imperfect repeats of about 70 base-pairs that are also present in front of other VSG genes, as shown by hybridization. The termini of three minor VSG 118-specific transcripts map within these repeats. The repeats have the potential to adopt non-B-DNA conformations, and could play a role in the recombination process that exchanges VSG genes in the expression site or, less likely, in pre-mRNA processing. Comparison of the DNA and the mRNA sequence has previously revealed that a terminal exon of 35 nucleotides is spliced onto the main body of the RNA. We show here that these 35 nucleotides are not in the transposed segment and they must, therefore, be contributed by the expression site. This argues persuasively that the transposition activates VSG gene expression by promoter addition rather than by a position effect.

Amino Acid Sequence↗
PubMed2000

JESAM: CORBA software components to create and publish EST alignments and clusters.

MOTIVATION: Expressed Sequence Tags (ESTs) are cheap, easy and quick to obtain relative to full genomic sequencing and currently sample more eukaryotic genes than any other data source. They are particularly useful for developing Sequence Tag Sites (STSs for mapping), polymorphism discovery, disease gene hunting, mass spectrometer proteomics, and most ironically for finding genes and predicting gene structure after the great effort of genomic sequencing. However, ESTs have many problems and the public EST databases contain all the errors and high redundancy intrinsic to the submitted data so it is often found that derived database views, which reduce both errors and redundancy, are more effective starting points for research than the original raw submissions. Existing derived views such as EST cluster databases and consensus databases have never published supporting evidence or intermediary results leading to difficulties trusting, correcting, and customizing the final published database. These difficulties have lead many groups to wastefully repeat the complex intermediary work of others in order to offer slightly different final views. A better approach might be to discover the most expensive common calculations used by all the approaches and then publish all intermediary results. Given a globally accessible database with a suitable component interface, like the JESAM software described in this paper, the creation of customized EST-derived databases could be achieved with minimum effort. RESULTS: Databases of EST and full-length mRNA sequences for four model organisms have been self-compared by searching for overlaps consistent with contiguity. The sequence comparisons are performed in parallel using a PVM process farm and previous results are stored to allow incremental updates with minimal effort. The overlap databases have been published with CORBA interfaces to enable flexible global access as demonstrated by example Java applet browsers. Simple cDNA supercluster databases built as alignment database clients are themselves published via CORBA interfaces browsable with prototypical applets. A comparison with UniGene Mouse and Rat databases revealed undesirable features in both and the advantages of contrasting perspectives on complex data. AVAILABILITY: The software is packaged as two Jar files available from: URL: http://corba.ebi.ac.uk/EST/jesam/jesam. html. One jar contains all the Java source code, and the other contains all the C, C++ and IDL code. Links to working examples of the alignment and cluster viewers (if remote firewall permits) can be found at http://corba.ebi.ac.uk/EST. All the Washington University mouse EST traces are available for browsing at the same URL.

Animals↗
PubMed2005

A structured interface to the object-oriented genomics unified schema for XML-formatted data.

Data management systems are fast becoming required components in many biology laboratories as the role of computer-based information grows. Although the need for data management systems is on the rise, their inherent complexities can deter the full and routine use of their computational capabilities. The significant undertaking to implement a capable production system can be reduced in part by adapting an established data management system. In such a way, we are leveraging the Genomics Unified Schema (GUS) developed at the Computational Biology and Informatics Laboratory at the University of Pennsylvania as a foundation for managing and analysing DNA sequence data in centromere research projects around Arabidopsis thaliana and related species. Because GUS provides a core schema that includes support for genome sequences, mRNA and its expression, and annotated chromosomes, it is ideal for synthesising a variety of parameters to analyse these repetitive and highly dynamic portions of the genome. Despite this, production-strength data management frameworks are complex, requiring dedicated efforts to adapt and maintain. The work reported in this article addresses one component of such an effort, namely the pivotal task of marshalling data from various sources into GUS. In order to harness GUS for our project, and motivated by efficiency needs, we developed a structured framework for transferring data into GUS from outside sources. This technology is embodied in a GUS object-layer processor, XMLGUS. XMLGUS facilitates incorporating data into GUS by (i) formulating an XML interface that includes relational database key constraint definitions, (ii) regularising traversal through that XML, (iii) realising automatic processing of the XML with database key constraints and (iv) allowing for special processing of input data within the framework for automated processing. The application of XMLGUS to production pipeline processing for a sequencing project and inputting the Arabidopsis genome into GUS is discussed. XMLGUS is available from the Flora website (http://flora.ittc.ku.edu/).

Chromosome Mapping↗
PubMed2003

Investigation of CGRP receptors and peptide pharmacology in human coronary arteries. Characterization with a nonpeptide antagonist.

Calcitonin gene-related peptide (CGRP), adrenomedullin (AM), and amylin are structurally related peptides mediating vasorelaxation in the coronary circulation possibly via CGRP receptors (subtypes 1 or 2). Functional CGRP1 receptors appear to consist of at least three different kinds of proteins: the calcitonin receptor-like receptor (CRLR), receptor-activity-modifying proteins (RAMPs) and the receptor component protein (RCP). No CGRP2 receptor has yet been cloned. Using reverse transcriptase - polymerase chain reaction, the presence of mRNA sequences encoding CRLR, RCP and RAMPs was demonstrated in human coronary arteries. Relaxant responses were studied on isolated segments of coronary arteries after precontraction with U46619 (9,11-dideoxy-11alpha,9alpha-epoxymethano-prostaglandin F(2alpha)). The human peptides alphaCGRP, AM, and amylin induced relaxation with mean pEC50 values of 8.6, 6.8, and 6.3 M, respectively. Preincubation with alphaCGRP(8-37) (10(-7) -10(-5) M) and a novel nonpeptide CGRP antagonist "Compound 1" (WO98/11128) (10(-7)-10(-5) M) caused a dose-dependent rightward shift of the concentration-response curves for alphaCGRP with pA(2) values of 7.0 and 7.1, respectively. Preincubation with alphaCGRP(8-37) (10(-6) M) and Compound 1 (10(-6) M) caused significant rightward shift of the concentration-response curves for AM and amylin as well with pK B values between 6.6 and 7.5. Preincubation with AM(22-52) had no antagonistic effect on the AM and amylin response, neither did diacetoamidomethyl cysteine CGRP cause any concentration dependent (10(-11)-10(-6) M) dilatation. In conclusion, mRNA for the components forming CGRP1 and AM receptors was detected in the human left anterior descending coronary arteries. alphaCGRP, AM, and amylin mediated vasorelaxation via the CGRP1 receptor. Compound 1 acted as a nonpeptide antagonist at the CGRP1 receptor and could thus become a tool for the study of CGRP-mediated functional responses in human tissue.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2000

Therapy with antisense TGF-beta1 oligodeoxynucleotides reduces kidney weight and matrix mRNAs in diabetic mice.

Inhibition of gene expression by antisense oligodeoxynucleotides (ODNs) relies on their ability to bind complementary mRNA sequences and prevent translation. The proximal tubule is a suitable target for ODN therapy in vivo because circulating ODNs accumulate in the proximal tubule in high concentrations. Because increased proximal tubular transforming growth factor- beta1 (TGF-beta1) expression may mediate diabetic renal hypertrophy, we investigated the effects of antisense TGF-beta1 ODN on the high-glucose-induced proximal tubular epithelial cell hypertrophy in tissue culture and on diabetic renal hypertrophy in vivo. Mouse proximal tubular cells grown in 25 mM D-glucose and exposed to sense ODN as control (1 microM) exhibited increased (3)[H]leucine incorporation by 120% and total TGF-beta1 protein by 50% vs. culture in 5.5 mM D-glucose. Antisense ODN significantly decreased the high-glucose-stimulated TGF-beta1 secretion and leucine incorporation. Continuous infusion for 10 days of ODN (100 microg/day) was achieved via osmotic minipumps in diabetic and nondiabetic mice. Sense ODN-treated streptozotocin-diabetic mice had 15.3% increase in kidney weight, 70% increase in alpha1(IV) collagen and 46% increase in fibronectin mRNA levels compared with nondiabetic mice. Treatment of diabetic mice with antisense ODN partially but significantly decreased kidney TGF-beta1 protein levels and attenuated the increase in kidney weight and the alpha1(IV) collagen and fibronectin mRNAs. In conclusion, therapy with antisense TGF-beta1 ODN decreases TGF-beta1 production and attenuates high-glucose-induced proximal tubular cell hypertrophy in vitro and partially prevents the increase in kidney weight and extracellular matrix expression in diabetic mice.

Animals↗
PubMedDate not supplied

Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by real-time polymerase chain reaction.

BACKGROUND CONTEXT: Serial analysis of gene expression during the course of intervertebral disc degeneration (IDD) could elucidate valuable insight into pathophysiology and provide a basis for identification of potential targets for the development of novel cellular- and gene-based therapies. However, very few previous studies described the changes in gene expression through the process of IDD using a suitable animal model. PURPOSE: To use a recently developed rabbit annular stab model and the technique of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify the change in expression of key rabbit-specific mRNA sequences encoding for selected extracellular matrix (ECM) products, catabolic, anabolic, and anti-catabolic factors in normal and stabbed discs. STUDY DESIGN: Gene expression analyses were performed to characterize a slowly progressive and reproducible animal model of IDD using real-time RT-PCR. METHODS: Twelve rabbits underwent an annular stab with a 16-gauge needle to the L2-L3, L3-L4, and L4-L5 discs, and three rabbits served as sham controls. Nucleus pulposus tissues were harvested from the stabbed discs at 3, 6, 12 and 24 weeks after confirmation of degenerative changes by magnetic resonance imaging (MRI) scan. Real-time RT-PCR was performed with the use of rabbit-specific primers for 1) extracellular matrix (ECM) component genes: collagen type Ia and IIa, and aggrecan; 2) catabolic genes: matrix metalloprotease-3 (MMP-3), inducible nitric oxide synthase (iNOS), and interleukin-1beta (IL-1beta); 3) anabolic growth genes: bone morphogenic protein-2, and -7 (BMP-2, -7), transforming growth factor-beta1 (TGF-beta1), and insulin-like growth factor-1 (IGF-1); and 4) anti-catabolic gene: tissue inhibitor of metalloprotease-1 (TIMP-1). These data were normalized to mRNA levels of glyceraldehyde phosphate dehydrogenase (GAPDH), a constitutively expressed gene. RESULTS: The MRI images confirmed progressive decline in the nucleus pulposus area of high T2 signal and in the signal intensity of the stabbed discs over the 24-week study period consistent with IDD. The ECM components, aggrecan and collagen type IIa mRNA levels had decreased markedly by week 3 and never recovered, whereas type Ia collagen mRNA gradually increased throughout course of degeneration. BMP-2, BMP-7 and IGF-1 mRNA were relatively decreased from weeks 3 to 6 but then increased at weeks 12 and 24 to end at a level near the preoperative level. The TIMP-1 expression fell dramatically to approximately one tenth of the preoperative level by week 3 and remained low throughout the degenerative process. The remaining results, including those from TGF-beta1 and the catabolic genes (MMP-3, IL-1beta, iNOS) demonstrated a double peak characteristic. The gene expression increased by week 3, decreased to a low level at weeks 6 and 12 and then had a second, late peak at 24 weeks. CONCLUSIONS: The gene expression profiles of ECM components and anabolic, catabolic, and anti-catabolic factors demonstrate many characteristics similar to the findings in human disc degeneration and suggest an inability of the intervertebral disc (IVD) to mount an early anabolic response to injury, thereby offering a possible explanation for the disc's lack of reparative capabilities. Catabolic genes are strongly up-regulated both early and late in degeneration, lending strong support to the hypothesis that an anabolic or catabolic imbalance plays a primary role in IDD. According to the resultant patterns, augmenting early production of BMP-2, BMP-7, IGF-1 or TIMP-1 by gene transfer techniques might possibly alter the progressive course of degeneration as seen in the stab model. The next step will be to transfer these therapeutic genes to regulate the biologic processes and ideally alter the progressive course of disc degeneration.

Animals↗
PubMed1999

Effects of anti-sense oligonucleotides directed toward dihydrofolate reductase RNA in mammalian cultured cells.

The effect of incubations with anti-sense phosphorothioate oligonucleotides directed toward sequences of dihydrofolate reductase (DHFR) RNA has been tested on Chinese hamster ovary cells. The selected targets were the 5'-untranslated region, the translational start, the splice sites and branch point of intron I and polyadenylation regions 1 and 3 of the DHFR RNA. To introduce the oligonucleotides, the cationic liposome DOTAP was used. The oligonucleotides most effective at causing cytotoxicity were ATNL and DTNL, both directed toward the translation-start site, at a range of concentrations between 1 and 4 microM. The minimum time for the oligonucleotide to exert its full cytotoxic effect was 3 days. Excess of oligonucleotide diminished the cytotoxic effect. Oligonucleotide uptake was monitored by the incorporation of [32P]- or fluorescein-labeled oligonucleotide and was found to depend on liposome and oligonucleotide concentrations and duration of incubation. Formation of in vitro complexes between the oligonucleotide and the liposome was also studied. Cytotoxicity was observed when the oligonucleotide was incubated with cell lines containing either the endogenous gene or co-transfected DHFR minigenes. Cell incubation with ATNL caused a time-dependent decrease in the levels of DHFR mRNA and enzymatic activity. Moreover, a cell line bearing amplification at the dhfr locus was equally affected by the action of ATNL. Human hepatoma cells were also affected by treatment with the counterpart of ATNL in the human DHFR mRNA sequence. Our results set the basis for a possible cancer therapy with anti-sense oligonucleotides using DHFR as the target.

Animals↗
PubMed1995

The effect of redox-related species of nitrogen monoxide on transferrin and iron uptake and cellular proliferation of erythroleukemia (K562) cells.

The iron-responsive element-binding protein (IRE-BP) modulates both ferritin mRNA translation and transferrin receptor (TfR) mRNA stability by binding to specific mRNA sequences called iron-responsive elements (IREs). The regulation of IRE-BP in situ could possibly occur either through its Fe-S cluster and/or via free cysteine sulphydryl groups such as cysteine 437 (Philpott et al, J Biol Chem 268:17655, 1993; and Hirling et al, EMBO J 13:453, 1994). Recently, nitrogen monoxide (NO) has been shown to have markedly different biologic effects depending on its redox state (Lipton et al, Nature 364:626, 1993). Considering this fact, it is conceivable that the NO group, as either the nitrosonium ion (NO+) or nitric oxide (NO+), may regulate IRE-BP activity by S-nitrosylation of key sulphydryl groups or via ligation of NO. to the Fe-S cluster, respectively. This hypothesis has been examined using the NO+ generator, sodium nitroprusside (SNP); the NO. generator, S-nitroso-N-acetylpenicillamine (SNAP); and the NO./peroxynitrite (ONOO-) generator, 3-morpholinosydnonimine hydrochloride (SIN-1). Treatment of K562 cells for 18 hours with SNP (1 mmol/L) resulted in a pronounced decrease in both the RNA-binding activity of IRE-BP and the level of TfR mRNA. In addition, Scatchard analysis showed a marked decrease in the number of specific Tf-binding sites, from 590,000/cell (control) to 170,000/cell (test), and there was also a distinct decrease in Fe uptake. Furthermore, SNP did not decrease cellular viability or proliferation. In contrast, the NO. generator, SNAP (1 mmol/L), increased RNA-binding activity of IRE-BP, the level of TfR mRNA, and the number of TfRs in K562 cells. Moreover, both SNAP (1 mmol/L) and SIN-1 (0.5 mmol/L) reduced cellular proliferation. The results are discussed in context of the possible physiologic role of redox-related species of NO in regulating iron metabolism.

Antioxidants↗
PubMed1980

A mouse immunoglobulin heavy chain deletion mutant: isolation of a cDNA clone and sequence analysis of the mRNA.

The mouse cell line IF2 secretes an immunoglobulin heavy chain lacking the CH1 domain. We have isolated and characterised a recombinant plasmid containing cDNA copies of the IF2 mutant mRNA. The cloned sequence extends from the nucleotides coding for amino acid 96 in the variable region through 100 nucleotides of untranslated region at the 3' end. The sequence of the cDNA insert reveals no discontinuity at the variable-hinge region junction, the site of the CH1 deletion. Experiments employing direct priming on the poly(A) tail of the IF2 heavy chain mRNA suggest that the 3' end of the cDNA clone (sequence C-C-C-T-G-C) is also the 3' end of the mRNA.

Animals↗