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PubMed2005

Synthesis of antisense oligonucleotides containing a photocleavable protecting group on a guanine base and their photoinduced duplex formation.

An oligonucleotide containing a photocleavable protecting group at a guanine base was synthesized to induce the duplex formation by photo-irradiation. Alpha-methyl-2-nitropiperonyl (MeNP) group was used for the photocleavable protecting group at O6 position of deoxyguanosine. The oligonucleotide containing MeNP group (MeNP-ODN:5'-dTTCTG(MeNP)TCTGT-3') was synthesized by phosphoramidite method. The MeNP group was found to be removable by UV irradiation at wavelength of 365 nm for 5 min in 98% yield. UV-melting temperature (Tm value) analysis indicated that the duplex of MeNP-ODN with the complementary RNA was significantly unstable compared with the unmodified DNA/RNA duplex (deltaTm = -25 degrees C). After UV irradiation at 365 nm, the Tm value of the mixture increased to the same as that of the unmodified duplex. These results suggest that the RNA binding ability of the MeNP-ODN can be induced by photocleavage of the MeNP group.

Benzodioxoles↗
PubMed2005

Synthesis and properties of photo-reactive antisense oligonucleotides containing 2'-O-psoralen-conjugated adenosine.

In order to selectively regulate mRNA having a point mutation, the photo-reactive antisense oligonucleotides were developed. Two types of photo-reactive oligonucleotides containing adenosine whose 2'-OH was modified with 4,5',8-trimethylpsoralen (psoralen) were synthesized (2'-Ps-oligo). One contains psoralen via a methylene linkage (2'-Ps-met), and the other via an amidomethylene linkage (2'-Ps-amd). 2'-Ps-oligos were then subjected to the photo-cross-linking reaction. 2'-Ps-met cross-linked to the complementary RNA and scarcely did to the RNA having a single mismatch base. Contrarily, 2'-Ps-amd did not cross-link to both RNA strands. These results suggest the structure of the linkage might affect the efficiency of the photo-cross-linking.

Adenosine↗
PubMed2005

Synthesis and properties of 2',4'-BNA(NC), a second generation BNA.

We have recently designed and synthesized a novel bridged nucleic acid analogue 2',4'-BNA(NC), bearing an N-O bridged structure, which furnished both higher duplex and triplex-forming abilities and sequence selectivity towards complementary RNA and/or DNA, respectively, and showed excellent resistance against nuclease degradation. Duplex and triplex-forming abilities were slightly higher or similar to those of 2',4'-BNA and nuclease resistance was as high as that of S-oligo.

Bridged-Ring Compounds↗
PubMed1995

The rat intrinsic factor gene: its 5'-upstream region and chief cell-specific transcription.

A DNA segment containing the 5'-upstream region and amino terminal reading frame of the gastric intrinsic factor gene was cloned from rat and its nucleotide sequence was determined. S1 mapping demonstrated that the transcription initiation site is located downstream of the second TATA-box sequence. Similar sequence motifs to those in the pepsinogen genes transcribed in gastric chief cells were found in the deduced sequence, suggesting that the rat intrinsic factor gene is transcribed in these cells. The genes for the intrinsic factor and its homologous protein transcobalamin I were apparently derived from a common ancestoral gene, since the positions of their intron insertions as well as the amino acid residues are conserved. Northern blot hybridization showed that the gene for the intrinsic factor is transcribed in the stomach but not detectably in the intestine, kidney, testis, brain, heart, liver, lung, or spleen. In situ hybridization using radioactive complementary RNA clearly indicated that the major transcription site in gastric glands is chief cells. Different locations of expression of intrinsic factor proteins in various mammals were observed previously using antibodies: in rat parietal cells and chief cells, in mouse chief cells, and in human parietal cells. The present results clearly demonstrated the intrinsic factor mRNA mainly in chief cells of adult rats, as in mice, suggesting that transcriptional regulation of the intrinsic factor gene is essentially the same in rodents.

Animals↗
PubMedDate not supplied

An unusual repetitive element from highly virulent isolates of Leptosphaeria maculans and evidence of its transfer to a weakly virulent isolate.

A 5,238-bp repetitive DNA element from a highly virulent isolate of Leptosphaeria maculans has been cloned and sequenced. The element is present in approximately 80 copies per haploid genome and hybridizes to every chromosome resolved by pulse field gel electrophoresis. The sequence is composed of 66% A+T and has numerous, very short, direct and inverted repeats. No RNA complementary to the element was detected in log phase cultures, and no open reading frames of significant length are present in the sequence. It has no structural similarity to other repetitive elements or significant homology to database sequences. We have designated the element LMR1. Southern blot hybridization indicated that the element is present in all isolates of L. maculans that are highly virulent to Brassica napus and B. rapa. The general structure of the element was conserved among isolates of different mating type, pathogenicity group, and geographic origin, as determined by both Southern blot analysis and primer-directed DNA amplification. LMR1 did not hybridize to DNA from weakly virulent strains of L. maculans, with the exception of one isolate. Phylogenetic analyses of restriction fragment length polymorphism and rDNA sequence indicated that the highly virulent and weakly virulent strains of L. maculans are not monophyletic. Therefore, the presence of the LMR1 element in a weakly virulent isolate may indicate that a rare transfer event has occurred. Surprisingly, the weakly virulent isolate that contains LMR1 is more pathogenic on B. napus and B. juncea than a similar isolate that lacks the element.

Ascomycota↗
PubMed2004

MTNR1A melatonin receptors in the ovine premammillary hypothalamus: day-night variation in the expression of the transcripts.

Melatonin regulation of reproductive functions in sheep is mediated by action in the premammillary hypothalamus (PMH). The aim of this study was to identify the high-affinity melatonin-receptor subtypes expressed in this structure. To achieve this, we used reverse transcription-polymerase chain reaction (RT-PCR) and developed in situ hybridization techniques (ISH). By using RT-PCR, we detected a band corresponding to the MTNR1A melatonin-receptor cDNA in the PMH as well as in the pars tuberalis (PT). On the opposite, MTNR1B melatonin-receptor transcripts were not detected using degenerate primers in any of the structures considered, confirming the lack of expression of this receptor subtype in sheep. The expression of MTNR1A mRNA was further confirmed in the PMH by ISH with a 35S-labeled ovine MTNR1A riboprobe. We next investigated the variation in the expression of MTNR1A mRNA between the end of the day and the end of the night (absence and presence of melatonin, respectively). MTNR1A transcript expression was greater at the end of the night than at the end of the day in the PMH. In contrast, MTNR1A mRNA expression was lower at the end of the night than at the end of the day in the PT. No significant variation in the MTNR1A mRNA expression was observed in a more dorsal hypothalamic area. Overall, these results show that MTNR1A transcripts are expressed in the ovine PMH and that their expression follows a diurnal rhythm, which is different from the pattern of expression observed in the PT.

Animals↗
PubMed1995

Prolactin receptor expression in rat spermatogenic cells.

To identify target cells of prolactin (PRL) in the male gonad, the expression of prolactin receptor (PRL-R) mRNA in adult rat testes was investigated by in situ hybridization using a digoxigenin-labeled cRNA probe. We also investigated PRL binding to testicular cells in vitro. Signals for PRL-R mRNA were detected not only in interstitial cells but also in spermatogenic cells. Although the reaction was positive in all phases of spermatogonia and spermatocytes, it disappeared in early round spermatids. No signals were detected in elongated spermatids or spermatozoa. The signal intensity varied among each phase of spermatogenic cells. PRL-R mRNA was expressed in all stages of the cycle of the seminiferous epithelium. PRL-R were detected on the surface of Leydig cells, Sertoli cells, all phases of spermatogonia and spermatocytes, elongated spermatids, and spermatozoa. Ovine PRL. did not bind to round spermatids. In Leydig cells, pachytene spermatocytes, and spermatozoa, PRL-R were observed in relatively large numbers. There were fewer receptors in other phases of spermatogenic cells. These results indicate that PRL-R mRNA expression is almost consistent with PRL binding sites except for elongated spermatids and spermatozoa, and suggest that PRL may have direct effects on spermatogenic cells.

Animals↗
PubMed1998

Cyclic and maturation-dependent regulation of follicle-stimulating hormone receptor and luteinizing hormone receptor messenger ribonucleic acid expression in the porcine ovary.

We sought to characterize the ovarian expression of mRNAs for the receptors for FSH (FSHr) and LH (LHr) at the different stages of the estrous cycle of the pig and to localize the receptor mRNAs to individual cell types in follicles and corpora lutea as a function of their developmental status. Northern blot analyses indicated that multiple FSHr and LHr mRNA transcripts occur on Days 0, 4, 7, 12, and 16 of the estrous cycle. In situ hybridization analyses revealed considerable cyclic variation in expression among small, medium, and large follicles. In small follicles of both immature prepubertal ovaries and mature adult ovaries at all days of the cycle studied, FSHr mRNA expression was strongly positive and was spatially restricted to granulosa cells. FSHr mRNA expression was strongly positive in granulosa cells of medium follicles but had declined in granulosa cells of mature follicles on Day 0. LHr expression in small follicles, in contrast, was spatially restricted to theca cells and was estrous cycle-dependent. LHr expression in theca cells was weakly positive in small follicles on Days 12, 0, and 4, was strongly positive in small follicles on Day 7 and in medium follicles on Days 12 and 16, and had declined in large mature follicles on Day 0. LHr expression in granulosa cells was negative in small follicles on Days 4 and 7, weakly positive in medium follicles on Days 12 and 16, and positive in large follicles on Day 0. In degenerating follicles, LHr mRNA was expressed weakly in the theca cells, and FSHr mRNA expression in granulosa cells was absent. In the corpus luteum, FSHr mRNA was not expressed, but LHr mRNA was expressed in some cells on Days 4 and 7, was expressed maximally on Day 12, and had declined partially by Day 16. These studies indicate that there is maturation-dependent regulation of FSHr and LHr expression in the adult porcine ovary. FSHr mRNA content decreases in parallel with FSHr protein content as follicles increase in size.

Animals↗
PubMed1998

Expression of mRNAs encoding BDNF and its receptor in adult rat hypothalamus.

We used a digoxigenin-UTP-labeled cRNA probe with in situ hybridization and Northern blot analysis to investigate the localization of brain derived neurotrophic factor (BDNF) mRNA and expression of its different transcripts in adult rat hypothalamus. As the BDNF gene is under the control of alternative multiple promoters, which provide tissue-specific gene expression, we studied whether these transcripts were expressed in adult hypothalamus. Our data revealed two novel sources of hypothalamic BDNF mRNA: the supraoptic and periventricular nuclei. In addition, we observed the expression of transcripts from exons, I, II and III as well as the presence of 1.6 and 4.2 kb BDNF mRNAs. Finally, our findings confirmed expression of mRNA encoding neurotrophins receptors in the hypothalamus.

Animals↗
PubMed2004

Higher viral load may explain the dominance of CRF02_AG in the molecular epidemiology of HIV in Ghana.

Phylogenetic analysis of Ghanaian plasma samples from 84 asymptomatic candidate blood donors and 150 AIDS patients revealed a 63% prevalence of HIV-1 CRF02_AG. HIV-1 viral load in both populations was quantified using real-time reverse transcriptase-polymerase chain reaction. In asymptomatic candidate blood donors infected with CRF02_AG, the viral load was significantly higher than in donors infected with other HIV-1 molecular forms. A higher viral load of CRF02-AG is compatible with higher infectivity and prevalence.

Ghana↗
PubMed1988

Quantification of renin mRNA in various mouse tissues by a novel solution hybridization assay.

A new solution hybridization assay was established for the measurement of renin mRNA. The assay makes use of a radioactively labelled renin complementary RNA as hybridization probe and allows rapid and sensitive detection of renin mRNA in amounts as low as 0.5 pg. With this assay it was possible to quantify renin mRNA in tissues with low-gene expression such as brain and heart as well as in testis, kidney and the submandibular gland (SMG) of mice. The concentrations in these organs were 0.03, 0.07, 1, 5.2 and 164 pg renin mRNA/micrograms RNA, respectively. These results are in agreement with Northern blotting experiments. The general applicability and ease of the solution hybridization assay described here should greatly improve the rapidity of mRNA measurements in future functional studies.

Animals↗
PubMed1991

Expression of endothelin-like peptide in the nervous system of the marine mollusk Aplysia.

The distribution of endothelin (ET)-like peptide immunoreactivity and mRNA in the nervous system of Aplysia californica was investigated by means of immunocytochemistry and in situ hybridization. Using specific antisera to human endothelin-1 (ET-1) and to the c-terminal peptide of human big endothelin-1 (22-38) (big ET-1), ET-like immunoreactivity was localized to various types of neurons. Immunoreactive nerve fibers for both antisera were prominent throughout the nervous system. Hybridization of radiolabeled complementary RNA probes for human ET-1 or rat endothelin (endothelin-3, ET-3) to paraformaldehyde-fixed, paraffin-embedded sections of various ganglia revealed the presence of a large number of labeled neurons. The number of labeled neurons was higher than those that were immunoreactive for the ET antisera. Preabsorption of the ET antisera with their respective synthetic peptide or hybridization of sections with the sense probes confirmed the specificity of the data. The present study shows that members of the endothelin family may be found in very distant phylogenetic organisms. This conservation suggests a possible role for this peptide as a neurotransmitter and/or neuromodulator in the central nervous systems of diverse species.

Animals↗
PubMed1997

E- and L-selectin adhesion molecules in musculocutaneous flap reperfusion injury.

Definition of the elements governing leukocyte adhesion to the microvascular endothelium may lead to new forms of treatment for reperfusion injury. The objectives of this study, employing a porcine latissimus dorsi flap reperfusion model, were (1) to characterize the expression of E- and L-selectin adhesion molecules and (2) to test for a possible benefit of E- and L-selectin blockade in the preceding experimental setting. In experiment 1, full-thickness biopsies were collected sequentially over an 8-hour ischemia and subsequent 6-hour reperfusion period. Immunocytochemistry was performed with monoclonal antibody EL-246, an antibody that crossreacts with both E- and L-selectin. In experiment 2, the binding of EL-246 to L-selectin on circulating porcine neutrophils was determined by flow cytometric analysis. In experiment 3, in situ hybridization was performed using complementary RNA probes for detection of endothelial E-selectin mRNA. In experiment 4, bilateral flaps were elevated in six pigs and subjected to 8 hours of arterial ischemia followed by 20 hours of reperfusion. Flaps on each animal were randomly assigned to receive either treatment with a continuous local intraarterial infusion of EL-246 (1 mg per flap) or solvent vehicle. Muscle and skin survivals were assessed by nitroblue tetrazolium and intravenous fluorescein staining techniques, respectively. Computer digitization permitted quantitation of relative tissue survival. In experiment 1, specific immunostaining of microvascular endothelium was achieved using EL-246. Greater-intensity staining was detected in reperfusion than in baseline or ischemic sections. In experiment 2, flow cytometric analysis indicated specific recognition by EL-246 of isolated peripheral porcine neutrophils (> 45 percent staining) as compared with an isotype-matched control antibody (< 3 percent staining). In experiment 3, in situ hybridization studies demonstrated an early ischemic up-regulation and later reperfusion downregulation of E-selectin mRNA during the reperfusion period. In experiment 4, administration of monoclonal antibody EL-246 afforded a significant augmentation in mean percentage survival of muscle (37.6 versus 18.7 percent, p = 0.015) and skin (48.6 versus 29.3 percent, p = 0.046). In conclusion, it was determined that E-selectin is expressed along the microvascular surface and is upregulated and subsequently downregulated during ischemia-reperfusion conditions. The monoclonal antibody EL-246 appears to recognize porcine L-selectin as well as E-selectin. Blockade of E/L-selectin-mediated leukocyte adhesion significantly reduces musculocutaneous flap reperfusion injury.

Animals↗
PubMed1996

Upregulation of IGF-I and collagen I mRNA in human atherosclerotic tissue is not accompanied by changes in type 1 IGF receptor or collagen III mRNA: an in situ hybridization study.

BACKGROUND: Immunoreactive insulin-like growth factor-I (IGF-I) has recently been localized to vascular smooth muscle cells in coronary atherectomy plaques, but it remains unclear whether these cells are the source of this growth factor. We therefore investigated the gene expression of this factor, and the expression of the genes for its receptor and two types of collagen known to be regulated by IGF-I, in vascular tissue samples from patients with atherosclerosis or restenosis. METHODS: Gene expression and localization were investigated by in situ hybridization, using 35S-labelled complementary RNA probes specific for IGF-I, its type a receptor, collagen I, and collagen III. The cellular composition of the tissue samples was determined by immunohistochemistry using antibodies specific for smooth muscle cells and macrophages. RESULTS: IGF-I and collagen I messenger RNAs were found in areas containing smooth muscle cells and macrophages, but collagen III and type 1 IGF receptor gene expression could not be detected in any tissue samples. CONCLUSION: IGF-I appears to be involved in the progression of the atherosclerotic plaque, even at an advanced stage, but preliminary data from two restenotic plaques indicate that it may not be involved in the later stages of restenosis.

Arteriosclerosis↗
PubMed1996

Simultaneous quantitation of cytokine mRNAs by reverse transcription-polymerase chain reaction using multiple internal standard cRNAs.

Cytokines produced in abnormal amounts or patterns contribute to many immunologically mediated human diseases. We describe a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure interleukin (IL)1-2, IL-4, and interferon-gamma (IFN-gamma) mRNAs within the sample. Internal standard cRNAs and native cytokine mRNAs are reverse transcribed and then amplified by PCR in the same reaction tubes to control for tube-to-tube variability in these reactions. In contrast to systems that use a single multigene internal standard cRNA, this method uses separate internal standard cRNAs for IL-2, IL-4, and IFN-gamma, allowing independent dosing of the internal standards, which reduces the number of tubes processed and the amount of starting mRNA required. Internal standards are produced from cytokine cDNAs by the insertion of short segments of DNA. The same oligonucleotide primers are used to amplify internal standard and native cytokine cDNAs. Each internal standard cDNA and its matching native cytokine cDNA are amplified with equal efficiency. The RT-PCR products of the internal standards and native cytokines are distinguished by size. This technique can detect a twofold difference in mRNA levels. Examples of using this technique to measure cytokine mRNAs in peripheral blood mononuclear cells and in bronchoalveolar lavage cells are given.

Bronchoalveolar Lavage Fluid↗
PubMed1998

Diagnostic implications of albumin messenger RNA detection and cytokeratin pattern in benign hepatic lesions and biliary cystadenocarcinoma.

Cytokeratin (CK) patterns and albumin messenger RNA (mRNA) are investigated in 24 patients with benign hepatic lesions (7 patients with focal nodular hyperplasia [FNH], 10 with hepatocellular adenomas [HA], 1 with biliary hamartoma, 4 with biliary cysts, 2 with cystadenomas) and in 8 patients with cystadenocarcinoma, a rare liver malignancy. The lesions and surrounding tissue of the hepatocytic components expressed CK 8 and 18 at immunohistochemistry, whereas the biliary elements evidenced CK 8 and 18 and CK 7 and 19. The albumin mRNA, as detected by in situ hybridization (ISH), revealed different distributions in the hepatocytes of FNH and HA. In the benign biliary lesions, the normal hepatocytes surrounding the tumors expressed albumin mRNA, whereas the biliary structures did not. Interestingly, in the cystadenocarcinomas, albumin mRNA was observed not only in the hepatocytes of residual parenchyma, but also in neoplastic bile duct cells lining the carcinomatous cysts; no signal was identified in the nonneoplastic biliary elements. This indicates that cystadenocarcinomas have a mixed biological phenotype and suggests they could arise either from pluripotent cells or from neoplastic cells that reacquire epigenetic features. Our results suggest two possible diagnostic applications for albumin ISH: on routine sections, it could represent an important tool for distinguishing between cystadenoma and cystadenocarcinoma; and on fine needle biopsy specimens, it could reduce uncertainty between FNH and HA.

Adenoma↗
PubMed2003

Ethanol sensitivity of recombinant homomeric and heteromeric AMPA receptor subunits expressed in Xenopus oocytes.

BACKGROUND: Ethanol is known to acutely inhibit AMPA receptor function, and sensitivity of AMPA receptors to ethanol is dependent on subunit composition in vivo and in vitro. A commonly used in vitro expression system for studying recombinant receptor subunits is the Xenopus laevis oocyte and two-electrode voltage-clamp electrophysiological recording. To date, ethanol sensitivity of injected receptor subunit complementary RNA (cRNA) has not been shown to be correlated with the actual expression of receptor subunits in oocytes. In this study, we compared ethanol sensitivity of homomeric and heteromeric AMPA receptor subunits microinjected into Xenopus oocytes and confirmed subunit expression in oocytes by immunoblot. METHODS: cRNAs coding for the "flop" type AMPA GluR1 or GluR3 (homomeric), GluR2/GluR3 (heteromeric combination), and GluR1/2/3 (heteromeric combination) were microinjected in equimolar amounts of 16 to 20 ng into oocytes, which were studied for their sensitivity to ethanol. Oocytes injected with cRNA for homomeric or heteromeric subunit combinations were homogenized and the expressed subunits quantified with anti-GluR1, anti-GluR2, and anti-GluR2/3 antibodies. RESULTS: Ethanol concentrations of 10 to 500 mM consistently inhibited currents activated in oocytes by 200 microM kainic acid. The expressed homomeric GluR1 receptor and heteromeric GluR1/2/3 receptor combination currents showed similar sensitivity to ethanol inhibition with half-maximal inhibition values of 170 +/- 12 mM and 176 +/- 8 mM, respectively. The expressed homomeric GluR3 receptor and heteromeric GluR2/3 receptor combination currents were differentially sensitive to ethanol inhibition with respective IC50 values of 238 +/- 9 mM and 338 +/- 16 mM. CONCLUSION: The expressed homomeric and heteromeric "flop" type AMPA receptors were differentially sensitive to ethanol, which may in part explain differential ethanol sensitivity in native neurons.

Animals↗
PubMed2005

Gene expression profiling in squamous cell carcinoma of the oral cavity shows abnormalities in several signaling pathways.

OBJECTIVES/HYPOTHESIS: To examine gene expression profiles in squamous cell carcinoma of the oral cavity (oral SCC) compared with histologically matched normal tissue. STUDY DESIGN: Fresh-frozen tissue was prospectively obtained from individuals undergoing surgical resections for oral SCC. METHODS: RNA was extracted from seven sets of oral SCC and matched normal tissue. Gene expression profiles were obtained by interrogation of Affymetrix Gene Chip Arrays with cRNA prepared from the tissue. Expression values were subjected to a paired t test. Genes that were judged to differ between oral SCC and normal tissue were annotated according to their name in the Affymetrix Netaffx database and according to their function as indicated by their Gene Ontology Consortium number. RESULTS: Of the 10,599 probe sets that were analyzed, 523 genes were abnormally expressed in SCC of the head and neck (P < or = .01), and 417 of these genes were abnormally expressed in all seven tumors in the same manner. Hierarchical clustering of the 121 genes that were abnormally expressed in cancerous relative to normal tissue (P < or = .001) showed that the tissue segregated into two groups consisting of normal and transformed tissue, as expected. The abnormal expression of two genes that were up regulated in oral SCC (ADAM 12 and PTHLH) and two genes that were down-regulated in SCCHN (EMP-1 and P11) were confirmed by real-time polymerase chain reaction and immunohistochemistry (ADAM 12) using additional sets of tissue. CONCLUSIONS: The data showed that oral SCC aberrantly express genes in cellular pathways related to proliferation, apoptosis, extracellular matrix degradation, adhesion, transforming growth factor-beta signaling, and transcription. Further work is needed to determine the role of these genes in the development and progression of oral SCC.

ADAM Proteins↗