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At least 811 records · Page 45Linked to original sources
PubMed1995

Participation of a proton-cotransporter, MCT1, in the intestinal transport of monocarboxylic acids.

A molecular mechanism for the intestinal monocarboxylic acid transport was characterized by using a proton/monocarboxylate transporter, MCT1, in Chinese hamster ovary (CHO) cells, first found by Garcia et al. (Cell, 76, 865-873, 1994). Northern blotting analysis showed that MCT1-isomers exist in the rat and rabbit intestinal enterocytes and Caco-2 cells. The expression of [14C]lactic acid uptake by Xenopus laevis oocytes injected with rabbit intestinal mRNA was reduced by hybridizing the mRNA with a MCT1 cDNA of CHO cells before microinjection used as the antisense DNA. [14C]Lactic acid uptake by CHO cells was pH dependent, saturable, stereospecific, and reduced in the presence of acetic acid, benzoic acid, S- and R-ibuprofen, S- and R-mandelic acid, nicotinic acid, pravastatin, propionic acid and valproic acid. In addition, several monocarboxylic acids were transported in pH-dependent and saturable manners. These results suggest that the intestinal MCT1-related protein contributes to a carrier-mediated absorption for organic weak acid compounds.

Animals↗
PubMed1996

Evidence that glutathione peroxidase RNA and manganese superoxide dismutase RNA bind the same protein.

Rat lung extract contains protein that binds to a cis element in the 3' untranslated region of glutathione peroxidase (GPx) mRNA; this region is located 200 bases downstream of the stop codon and 77 bases downstream of the selenocysteine insertion sequence. GPx mRNA-binding protein (GPx-BP) has the following characteristics in common with Mn superoxide dismutase mRNA-binding protein (MnSOD-BP): 1. RNA-binding activity is redox-sensitive; free sulfhydryl groups on the protein are required for binding. 2. RNA-binding activity is enriched in a 130,000 x g supernatant fraction and is inhibited by RNA in the polysomal fraction. 3. The MnSOD and GPx cis elements compete with each other for protein binding. 4. UV crosslinking studies with each probe reveal a [32P]-labeled protein of the same apparent molecular mass, 56 kDa. These observations provide evidence that MnSOD and GPx RNAs bind the same protein.

Animals↗
PubMed2000

Differential expression of sense and antisense transcripts of the mitochondrial DNA region coding for ATPase 6 in fetal and adult porcine brain: identification of novel unusually assembled mitochondrial RNAs.

The mammalian mitochondrial genome is a double-stranded circular DNA molecule, which is transcribed from both strands as polycistronic RNAs, which are further processed to yield the mature polyadenylated mRNAs, rRNAs and tRNAs. We compared the gene expression patterns of foetal and adult porcine brains and identified a sequence tag from the ATPase 6 region of the mitochondrial genome which, in adult brain, was more abundant in the sense (H-strand) form, but, in foetal brain, more abundant in the antisense form (L-strand). By means of solution hybridisation/S1 nuclease protection assay, Northern blotting, and PCR based techniques, we demonstrated that the ATPase 6 region of the porcine mitochondrial genome is transcribed as co-existing, stable sense and antisense RNAs. Furthermore, we identified sense and antisense transcripts from this region consisting of inversely assembled fragments joined together at a direct repeat of 7 nucleotides. Our results suggest that transcription and post-transcriptional processing of mitochondrial RNAs are much more complex than presently thought.

Adenosine Triphosphatases↗
PubMed2002

DNA microarray analysis of transforming growth factor-beta and related transcripts in nasal biopsies from patients with allergic rhinitis.

Decreased activity of anti-inflammatory cytokines like transforming growth factor (TGF)-beta may contribute to allergic inflammation. In vivo effects of TGF-beta-effects are difficult to infer from local concentrations, since TGF-beta-effects depend on a complex system of regulatory proteins and receptors. Instead the effects of TGF-beta might be inferred by examining TGF-beta-inducible transcripts. In this study DNA microarrays were used to examine local expression of TGF-beta, TGF-beta-regulatory and -inducible transcripts in nasal biopsies from patients with symptomatic allergic rhinitis and healthy controls. In addition, nasal fluids were analysed with cytological and immunological methods. Nasal fluid eosinophils, albumin, eosinophil granulae proteins and IgE, but not TGF-beta, were higher in patients than in controls. DNA microarray analysis of nasal mucosa showed expression of transcripts encoding TGF-beta, TGF-beta-regulatory proteins and -receptors at variable levels in patients and controls. By comparison, analysis of 28 TGF-beta-inducible transcripts indicated that 23 of these had lower measurement values in patients than in controls, while one was higher, and the remaining four were absent in both patients and controls. In summary, TGF-beta and a complex system of regulatory genes and receptors are expressed in the nasal mucosa. Low expression of TGF-beta-inducible transcripts may indicate decreased TGF-beta activity in allergic rhinitis. DNA microarray analysis may be a way to study cytokine effects in vivo.

Adolescent↗
PubMed1993

Inhibition of differentiation in P19 embryonal carcinoma cells by the expression of vectors encoding truncated or antisense EGF receptor.

Murine embryonal carcinoma cells do not express detectable cell-surface epidermal growth factor receptors (EGF-R) but after 2 days of differentiation induced by retinoic acid (RA) increasingly express mRNA and protein encoded by the EGF receptor gene (Joh et al., Cell Growth and Differentiation 3, 315, 1992). The effect on morphology, growth, and differentiation of the introduction of expression vectors that produce either a truncated, kinase-negative mouse EGF receptor or an antisense mRNA was studied in P19 embryonal carcinoma (EC) cells before and after differentiation. The presence of either construct should lead to the reduction of EGF-R expression by either the dominant negative effects of a truncated protein or the inhibition of endogenous EGF-R mRNA production/translation by complementary RNA, respectively. Cells were cotransfected with the bacterial neomycin resistance gene and constitutively expressing clones were selected with G418. The cytomegalovirus LTR promoter/enhancer was found to be very inefficiently activated in P19 EC cells. After RA addition, changes in gene expression included induction of both the exogenous truncated constructs and endogenous EGF-R. Differentiation was gauged by the expression of tissue-type plasminogen activator and intermediate filament protein markers of neural tissues, as well as EGF-R. The expression of 120-kDa truncated EGF-Rs was high in four clones, but all 10 clones examined had diminished abilities to differentiate after RA induction compared to four control cell lines. Similarly, the majority of antisense transfected clones was unable to differentiate normally. The results indicate that the reduced expression of EGF-Rs in differentiating EC cells inhibits the rate, frequency, and extent of differentiation after RA induction. We conclude that the expression of EGF-Rs plays a role in the stimulation of differentiation and we speculate that the mechanism involves the tyrosine kinase activity of the receptor.

Animals↗
PubMed1996

A ribonucleotide reductase inhibitor, MDL 101,731, induces apoptosis and elevates TRPM-2 mRNA levels in human prostate tumor xenografts.

MDL 101,731, (E)2'-fluoromethylene-2'-deoxycytidine, is an irreversible inhibitor of ribonucleotide diphosphate reductase and causes regression of human tumors in nude mouse models. Messenger RNA levels for testosterone-repressed prostatic message-2 (TRPM-2), a transcript that increases in human tumor xenografts undergoing programmed cell death, were analyzed by in situ hybridization. Xenografts derived from a human prostate tumor cell line (PC-3) regressed following treatment with MDL 101,731 and the relative levels of TRPM-2 mRNA increased up to threefold in drug-treated animals. Apoptosis in the tumor xenografts was further indicated by in situ labeling of DNA strand breaks by incorporation of biotinylated-dUTP with terminal deoxynucleotidyl transferase. In vitro, PC-3 cells incubated with MDL 101,731 showed evidence of apoptosis based on flow cytometry and DNA laddering. These data support the hypothesis that MDL 101,731 stimulates programmed cell death in regressing PC-3 xenografts.

Animals↗
PubMed1998

Regulation of semaphorin III/collapsin-1 gene expression during peripheral nerve regeneration.

The competence of neurons to regenerate depends on their ability to initiate a program of gene expression supporting growth and on the growth-permissive properties of glial cells in the distal stump of the injured nerve. Most studies on intrinsic molecular mechanisms governing peripheral nerve regeneration have focussed on the lesion-induced expression of proteins promoting growth cone motility, neurite extension, and adhesion. However, little is known about the expression of intrinsic chemorepulsive proteins and their receptors, after peripheral nerve injury and during nerve regeneration. Here we report the effect of peripheral nerve injury on the expression of the genes encoding sema III/coll-1 and its receptor neuropilin-1, which are known to be expressed in adult sensory and/or motor neurons. We have shown that peripheral nerve crush or transection results in a decline in sema III/coll-1 mRNA expression in injured spinal and facial motor neurons. This decline was paralleled by an induction in the expression of the growth-associated protein B-50/GAP-43. As sema III/coll-1 returned to normal levels following nerve crush, B-50/GAP-43 returned to precrush levels. Thus, the decline in sema III/coll-1 mRNA coincided with sensory and motor neuron regeneration. A sustained decline in sema III/coll-1 mRNA expression was found when regeneration was blocked by nerve transection and ligation. No changes were observed in neuropilin-1 mRNA levels after injury to sensory and motor neurons, suggesting that regenerating peripheral neurons continue to be sensitive to sema III/coll-1. Therefore we propose that a decreased expression of sema III/coll-1, one of the major ligands for neuropilin-1, during peripheral nerve regeneration is an important molecular event that is part of the adaptive response related to the success of regenerative neurite outgrowth occurring following peripheral nerve injury.

Animals↗
PubMed1996

Structure of the human laminin gamma 2 chain gene (LAMC2): alternative splicing with different tissue distribution of two transcripts.

We have determined the structure of the human laminin gamma 2 chain gene (LAMC2), which is mutated in some patients with junctional epidermolysis bullosa. Eight lambda phage clones isolated from a genomic library and three subgenomic lambda phage clones made from a plasmid artificial chromosome clone spanned 75 kb, including the 55-kb gene. The LAMC2 gene contains 23 exons and is structurally highly homologous with the 28-exon LAMC1 gene (Kallunki et al., 1991, J. Biol. Chem. 266: 221-228), with 16 exons having identical sizes in the two genes. The gene analysis demonstrated that two previously described different size gamma 2 chain cDNAs (Kallunki et al., 1992, J. Cell Biol. 119: 679-693) are the result of alternative splicing. The longer gamma 2 chain is formed by using the coding sequence of the last exon 23, while the shorter gamma 2* chain is formed by using only 22 exons, together with part of the 5' end of intron 22. The two mRNAs were shown to have different expression patterns in 17-week-old human embryonic tissues, with the longer gamma 2 chain transcript strongly expressed in epithelia of all tissues studied, while distinct expression of the shorter gamma 2* chain mRNA was observed only in the cerebral cortex, in lung, and in distal tubules of the kidney.

Alternative Splicing↗
PubMed2001

Spatially restricted expression of regulators of G-protein signaling in primary olfactory neurons.

The intracellular signal transduction machinery of heterotrimeric G-protein coupled odorant and putative pheromone receptors converts odorous information into a cellular response. We have investigated for the presence of 18 members of the family termed "regulators of G-protein signaling" (RGS) in primary olfactory sensory neurons of the main as well as the accessory (vomeronasal) system of the mouse. Unexpectedly, expression of a few RGS members show spatial restrictions correlating with the patterns described for G-protein coupled receptors in these two types of olfactory neurons. RGS3 was selectively coexpressed with the Galphai2 G-protein subunit in a subpopulation of vomeronasal neurons. The mutually exclusive spatial extents of RGS9 and RGSZ1 expression in main olfactory neurons corresponded precisely to that of certain odorant receptor zones. This renders these RGS members the first described intracellular signal transduction components with a potential role in the spatially organized sensory coding in the main olfactory system.

Animals↗
PubMed1996

A rapid and convenient method to prepare DIG-labelled RNA probes for use in non-radioactive in situ hybridization.

We describe here the use of PCR-generated templates incorporating T3 polymerase sites in order to prepare digoxigenin (DIG)-labelled cRNA probes against any gene of known sequence. This method was applied to the preparation of probes specific for chicken glyceraldehyde-3-phosphate dehydrogenase messenger RNAs and we demonstrate that such probes can be used for in situ hybridization (ISH). This technique therefore represents a rapid and convenient means to prepare DIG-labelled cRNA probes for use in a non-radioactive ISH. It adds speed and convenience of probe preparation to the previously described advantages of non-radioactive detection techniques.

Animals↗
PubMed1996

Competitive reverse transcription/polymerase chain reaction for the quantification of p53 and mdm2 mRNA expression.

Wild-type p53 (wtp53) is a tumour suppressor gene involved in cell cycle regulation. The mdm2 protein can complex with the p53 protein and influence its function as a regulator of cell growth. To detect and quantify wtp53 and mdm2 mRNA expression, we established the competitive reverse transcription/polymerase chain reaction for these genes and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The target RNA differed from the competitor cRNA by having 183 bp, 205 bp and 173 bp deletions for p53, mdm2 and GAPDH, respectively. Target RNA and known concentrations of competitor cRNA were co-reverse transcribed and co-amplified with the same primers. Target cDNA and the corresponding competitor cDNA were amplified at the same efficiency.

Endothelium, Vascular↗
PubMed1997

Amplifiable hybridization probes containing a molecular switch.

In order to reduce background signals in Q beta replicase-mediated bioassays, a target-dependent probe amplification strategy has been proposed that utilizes recombinant RNA hybridization probes that contain an inserted molecular switch. A molecular switch is an internal region of the probe that undergoes a conformational change when the probe hybridizes to its target. We investigated whether non-hybridized probes (which cause background signals) could be selectively destroyed by incubating the probe-target hybrids with ribonuclease III, which should cleave the non-hybridized probes and leave the hybridized probes intact. Two problems with this assay design were observed. First, ribonuclease III cleaved probe-target hybrids non-specifically when the target was an RNA, thereby destroying all of the bound probes. And second, the expected conformational change in the molecular switch did not occur when the probes were bound to their targets, apparently because the hairpin stem formed by the molecular switch was too long. Although these results demonstrated that the original assay design could not work, they provided insights that have led to better designs for target-dependent amplification assays. In these assays, the probes will be DNA molecules containing short-stemmed molecular switches. Non-hybridized probes will be selectively destroyed by incubation with a restriction endonuclease.

Blotting, Northern↗
PubMed1997

A rapid and sensitive method for non-isotopic quantitation of HIV-1 RNA using thermophilic SDA and flow cytometry.

Thermophilic strand displacement amplification (tSDA) is an isothermal DNA amplification technique that proceeds at 55-60 degrees C using both a thermostable restriction enzyme and a DNA polymerase. A modification of this system has been developed that allows the simultaneous amplification and detection of a DNA target by the addition of a detector probe to the reaction. This tSDA system has been further modified into a flow cytometry-based, bead capture assay for quantitation of HIV-1 RNA. A biotinylated capture probe and digoxygenin-dUTP have been incorporated into the tSDA reaction. The resulting double labelled amplicons are captured on strepavidin beads, and a fluorescent signal is generated on the beads by staining with fluorescent anti-digoxygenin antibody. The assay has a linear dynamic range of three orders of magnitude with a lower detection limit at 250 HIV-1 RNA molecules.

Biotinylation↗
PubMed1999

Quantification of iNOS mRNA with reverse transcription polymerase chain reaction directly from cell lysates.

Inducible nitric oxide synthase (iNOS) is a member of a family of primary inflammatory response genes. Quantitative measurement of iNOS mRNA levels is important for the study of gene expression of this enzyme during the process of inflammation. We report here a method for quantitative measurement of iNOS mRNA levels with rtPCR directly from cells lysed with a single step phenol/chloroform/ether extraction. Using a mouse macrophage cell line, J774.2, which expresses iNOS mRNA upon LPS + IFN-gamma treatment as the model, the effects of the extraction on iNOS mRNA recovery and cytosolic RNase removal have been studied. The cells are lysed and RNases denatured and removed by phenol/chloroform extraction. Trace amounts of the phenol partitioned in the samples are then removed by ether extraction. After the extraction, the samples can be used directly for reverse transcription and PCR without further purification of RNA. The recovery of specific mRNA is not affected by the extraction procedure and externally added iNOS cRNA shows no degradation by the extracted cell lysates. Measurement of iNOS mRNA with this procedure is linear using serially double-diluted cells in the range from 94 to 6000 cells. The efficiencies of rtPCR of iNOS wild-type and deletion cRNAs are also compared in our study. By controlling the molecular size of the deletion construct to within 10% of that of the wild type and maintaining PCR cycling below 25 cycles, the rtPCR efficiencies of iNOS wild type and deletion are identical. The detection of rtPCR products is enhanced by hybridization with specific probes. Under these conditions, iNOS mRNA concentration can directly be calculated from the internal standard in each tube without a standard curve. We conclude that our procedure provides an accurate method for quantitative measurement of iNOS mRNA from limited amount of cells without complete RNA isolation.

Animals↗
PubMed2002

Local synthesis of proteins at synaptic sites on dendrites: role in synaptic plasticity and memory consolidation?

Long-lasting forms of memory are thought to be mediated by modifications in synapses that are induced by particular patterns of activity, take time to be established (consolidated), and involve new gene expression. The molecular mechanisms underlying such long-lasting synaptic modifications remain to be defined. Here, we focus on new findings regarding synapse-specific gene expression and especially the intracellular transport and synaptic targeting of the mRNA for a recently identified immediate early gene called activity-regulated cytoskeleton-associated protein (Arc). Arc mRNA is transported into dendrites after episodes of neuronal activation. The newly synthesized Arc mRNA localizes selectively at synapses that have experienced particular patterns of activity, and this targeting depends on NMDA receptor activation. Arc protein is assembled into the postsynaptic junction, and Arc expression is strongly induced by brief behavioral experiences in select populations of neurons. These features of Arc induction and trafficking reveal novel cellular mechanisms that are well suited to mediate long-term synapse-specific modifications. We will review findings from other laboratories that both LTP and memory are disrupted when Arc induction is blocked, and we will discuss the possibility that Arc protein may play a key role in activity-dependent synaptic modification.

Cytoskeletal Proteins↗
PubMed1994

Characterization and detection of sc4: a sixth gene encoded by sonchus yellow net virus.

The nucleotide sequence of a sixth gene (sc4) of the plant rhabdovirus sonchus yellow net virus (SYNV) was determined from viral genomic and poly(A)+ cDNA clones. The sc4 gene is 1196 nucleotides (nt) and has an open reading frame of 972 nt that is capable of encoding a protein of 324 amino acids. Primer extension analyses of poly(A)+ RNA isolated from infected plants indicate that the 5' end of the sc4 mRNA corresponds to nucleotide 2840, relative to the 3' end of the minus-sense genomic RNA and extends to nucleotide 4035. A 43-nt untranslated leader sequence precedes the predicted first AUG codon and a 181-nt untranslated sequence follows the translational stop codon. This gene is similar to the other SYNV genes in that it is flanked on each side by a conserved gene junction sequence. Polyclonal antibodies raised to an sc4 fusion protein react with a 37-kDa protein in virus-infected plants that is close to the predicted size of the sc4 protein. Western blot analyses of cellular fractionations from infected plants show that sc4 is membrane associated and sucrose density gradient analyses demonstrate that sc4 sediments in the same fractions as SYNV virions. Analysis of the sc4 open reading frame reveals that 16% of the amino acids are serine or threonine residues and that the protein has four potential consensus casein kinase II phosphorylation sites. The deduced amino acid sequence of sc4 also contains a motif related to alpha amylases and aspartic proteases. This completes the sequence determination of the 13,720-nt SYNV genome.

Amino Acid Sequence↗
PubMed1995

Expression of cytokeratin-mRNAs in squamous-cell carcinoma and balloon-cell formation of human oesophageal epithelium.

Using digoxigenin-labelled cRNA probes, relationships between morphological characteristics and in situ hybridization for cytokeratin (CK)-mRNAs were analysed in cases of squamous-cell carcinoma of variable differentiation and in balloon-cell formation within the oesophageal mucosa. The present results were correlated to our previous findings on normal oesophageal epithelium. Our results from in situ hybridization study on oesophageal squamous-cell carcinoma provide strong evidence that changes in CK expression occur with differences in malignant potential. Cells of poorly differentiated carcinoma lose an ability to produce CK-mRNAs characteristic of their normal progenitor cells. Moderately differentiated and, still more pronounced, well differentiated carcinoma cells retain an ability to produce CKs characteristic of their tissue of origin (CK 6, CK 14, CK 15 and CK 19). Furthermore, well differentiated carcinoma cells may also gain an ability to synthesize new types of CKs that are not characteristic of the normal oesophageal epithelium (CK 8 and CK 18 characteristic of most simple epithelia, and CK 10 characteristic of keratinizing epithelia). Moreover, some oesophageal CK-genes are expressed in an obviously higher amount (CK 6, CK 14, and CK 19), but the expression of genes coding for the oesophageal differentiation-related CKs (CK 4 and CK 13) is obviously decreased or apparently lost. At the interface zone, observed in sections of well differentiated carcinomas, CK 8 and CK 18 mRNA were expressed in intermediate cell layers, and the centrally located cell layers were found positive for CK 10 mRNA. These findings largely extend the existing results from immunoblotting and immunohistochemical studies. The reduced or non-detectable expression of oesophageal differentiation-related CK-mRNAs (CK 4 and CK 13) on the appearance of balloon cells, suggests molecular changes that may be a marker for pathological progression. In addition, the abundant expression of CK 6 and CK 14 mRNA within areas of balloon-cell formation showing basal hyperplasia, and the higher expression of CK 19 in comparison with normal epithelium, points rather to de-differentiation than to normal vertical differentiation of the oesophageal epithelium. Whether CK-mRNAs can be used as biomarkers for evaluation of oesophageal pathologies remains to be further elucidated.

Carcinoma, Squamous Cell↗
PubMed1993

A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes.

We have developed a simple non-radioactive in situ hybridization procedure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of various transcripts present at a wide range of expression levels in the central nervous system. Cerebellar hybridization signals for transcripts estimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and low (inositol polyphosphate-5-phosphatase) levels of abundance are demonstrated as examples. The sensitivity and cellular resolution were significantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protocol can be combined with immunocytochemistry as demonstrated for glial fibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are described in detail.

Animals↗