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At least 991 records · Page 55Linked to original sources
PubMed1995

Differential targeting to the plasma membrane of the Torpedo 15-kDa proteolipid expressed in oocytes.

Xenopus laevis oocytes were injected with poly(A)+ RNAs extracted from the electric lobes of Torpedo marmorata, which contain a homogeneous population of cholinergic neurons. These primed oocytes were able to synthesize acetylcholine and to release the neurotransmitter in a calcium-dependent manner. Fractionation of oocyte membranes as well as immunofluorescence experiments showed that the 15-kDa proteolipid, a common subunit of the vacuolar H(+)-ATPase and of a presynaptic membrane protein capable of calcium-dependent acetylcholine translocation called the mediatophore, was located at the oocyte plasma membrane. In contrast, oocytes injected with separate transcripts encoding the 15-kDa proteolipid and choline acetyltransferase were unable to release acetylcholine in spite of an equivalent acetylcholine content and a higher level of 15-kDa proteolipid expression. We observed by immunofluorescence that under these conditions, the 15-kDa proteolipid was expressed in granular cytoplasmic membranes, which were then identified as being Golgi vesicles by cell fractionation. The striking difference in the distribution of the 15-kDa proteolipid expressed in oocytes primed with Torpedo electric lobe mRNA as compared with that seen in oocytes injected with the cRNA alone suggests that another protein endogenous to the electric lobe may be implicated in the localization of the 15-kDa proteolipid at the plasma membrane. Moreover, such a targeting mechanism could contribute to the capacity of electric lobe mRNA-injected oocytes to release acetylcholine.

Acetylcholine↗
PubMed1996

Human astrocytoma cells (U-87 MG) exhibit a specific substance P binding site with the characteristics of an NK-1 receptor.

To investigate substance P (SP) receptors on an established human astrocytoma cell line (U-87 MG), [3H][Sar9,Met(O2)11]-SP, a selective SP receptor agonist, was used to identify and characterize the cell membrane binding sites for SP. SP receptor mRNA was examined by solution hybridization analysis, and the existence of SP binding protein on the surface of membranes was evaluated by flow cytometry using an anti-SP binding protein antibody. In U-87 MG and U-373 MG RNA preparations, transcripts were identified that corresponded to both mature and partially spliced receptor forms. In U-87 MG cell membrane-enriched preparations, the binding of [3H][Sar9,Met(O2)11]-SP was found to be time and cell number dependent, specific, saturable, and of high affinity. Equilibrium binding analysis revealed a single class of binding sites with an apparent KD of 1.15 +/- 0.15 nM and a Bmax of 108 +/- 9.8 fmol/mg of protein. [3H][Sar9, Met(O2)11]-SP binding was basically not influenced by addition of mono (Na+, Li+) or divalent (Mg2+, Mn2+, Ca2+) cations; only high doses of divalent cations decreased the binding. GTP and guanylyl-5'-imidodiphosphate, but not GDP and GMP, reduced the Bmax without changing the affinity of [3H][Sar9,Met(O2)11]-SP. We also examined the effects of pretreatment with three lectins [concanavalin A (con A), wheat germ agglutinin (WGA), and Lens culinaris agglutinin (LCA)] to determine the nature of carbohydrate chains on the U-87 MG cell. Of three lectins analyzed for effects on agonist binding, WGA and LCA had an inhibitory effect, whereas con A was ineffective. These results suggest that SP receptors on the human astrocytoma cell line U-87 MG have either a biantennary complex-type or a high mannose-type of carbohydrate chain and may be regulated by GTP-binding protein(s).

Astrocytoma↗
PubMed2000

Effect of stress exposure on the activation pattern of enkephalin-containing perikarya in the rat ventral medulla.

We examined the effects of acute and chronic psychogenic stress on the activation pattern of enkephalin-containing perikarya in the rat ventrolateral medulla. Rats allocated to the chronic stress groups were subjected to 90 min of immobilization for 10 days. On the 11th day, the chronically stressed rats were exposed to homotypic (90-min immobilization) or to heterotypic but still psychogenic (90-min immobilization coupled to air jet stress) stress. The acute stress group was subjected once to an acute 90-min immobilization. For each group, the rats were anesthetized either before stress (time 0) or 90, 180, and 270 min after the onset of stress. Brain sections were then processed using immunocytochemistry (Fos protein) followed by radioactive in situ hybridization histochemistry (enkephalin mRNA). Following immobilization, the acute group displayed a marked increase in the number of activated enkephalin-containing perikarya within the paragigantocellularis and lateral reticular nuclei. This level of activation was sustained up to 180 min following the onset of the immobilization stress and had returned to baseline levels by 270 min from the initiation of the stress. However, this stress-induced activation of enkephalin-containing perikarya of the ventrolateral medulla was not seen following either homotypic or heterotypic stress in the chronically stressed group. These results provide evidence that enkephalin-containing perikarya of the ventrolateral medulla may constitute a potential circuit through which they regulate some aspect of the stress responses. Conversely, this enkephalinergic influence from the ventrolateral medulla was shown to be absent following chronic stress exposure. This would suggest a decrease in enkephalin inhibitory input originating from the ventrolateral medulla, thereby allowing a neuroendocrine and/or autonomic response to chronic stress.

Acute Disease↗
PubMed2001

Absolute quantification of AMPA receptor subunit mRNAs in single hippocampal neurons.

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit (GluR1-4) mRNAs expressed by single neurons in rat hippocampal cultures were quantified by single-cell RT-PCR using an internal standard RNA after whole-cell patch-clamp recording. The internal standard RNA, derived from GluR2 with a single nucleotide substitution, was reverse-transcribed and PCR-amplified with the same efficiency as GluR1-4 mRNAs. The mean mRNA numbers harvested in vitro from pyramidal-like neurons on day 9 were 1150 +/- 324 molecules of GluR1, 1080 +/- 273 molecules of GluR2, 100 +/- 20 molecules of GluR3, and 50 +/- 10 molecules of GluR4 (mean +/- SEM, n = 12). In a non-pyramidal neuronal population that expresses AMPA receptors characterized by high Ca(2+) permeability, the numbers of GluR1, GluR3 and GluR4 mRNA molecules harvested per cell were 354 +/- 64, 25 +/- 17 and 168 +/- 36, respectively (n = 8). The GluR2 mRNA was not detected in this cell type. The calculated ratio of AMPAR mRNA molecules per total mRNA molecules was 1/240 in pyramidal-like neurons (1/500 for GluR2), being in the range obtained with total RNA from rat forebrain and cerebellum (1/170 and 1/380, respectively). Finally, our results indicated that the proportion of GluR1-4 mRNA located in neurites reached approximately 60% in pyramidal-like neurons. However, we found no evidence of preferential subcellular distribution of a given subunit.

Animals↗
PubMedDate not supplied

[The peptide nucleic acids (PNAs): "high-tech" probes for genetic and molecular cytogenetic investigations].

The peptide nucleic acids (PNAs) constitute a remarkable new class of synthetic nucleic acids analogs, in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by amine bonds and to which the nucleobases are fixed. This structure gives to PNAs the capacity to hybridize with high affinity and specificity to complementary RNA and DNA sequences, and a great resistance to nucleases and proteinases. Originally conceived as ligands for the study of double stranded DNA, the unique physico-chemical properties of PNAs have led to the development of a large variety of research and diagnostic assays, including antigene and antisense therapy and genome mapping. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic polymorphisms and mutations or capturing nucleic acids. Over the last few years, the use of PNAs has proven its powerful usefulness in cytogenetics for the rapid in situ identification of human chromosomes and the detection of aneuploidies. Recent studies have reported the successful use of chromosome-specific PNA probes on human lymphocytes, amniocytes, spermatozoa as well as on isolated oocytes and blastomeres. Muticolor PNA protocols have been described for the identification of several human chromosomes, indicating that PNAs could become a powerful tool for in situ chromosomal investigation.

Cytogenetics↗
PubMed2003

Menstrual phase-dependent gene expression differences in periurethral vaginal tissue from women with stress incontinence.

OBJECTIVE: The mechanical stability of the genitourinary tract is dependent on intact collagen fibers that support the bladder neck, urethra, and pelvic organs. We hypothesize that genetic differences in collagen metabolism may contribute to stress urinary incontinence. Because sex hormones have substantial influence on the female lower urinary tract throughout adult life, we investigated the gene expression of vaginal tissue of women with stress incontinence compared with women with no stress incontinence in the proliferative phase of the menstrual cycle. STUDY DESIGN: Quantitative competitive polymerase chain reaction was used to verify that the gene expressions were similar between periurethral vaginal tissue and pelvic ligamentous tissue. Labeled complementary RNA was obtained from periurethral vaginal tissue in five pairs of age- and menstrual phase-matched, premenopausal women with and without stress urinary incontinence. The vaginal tissues were then hybridized on HuGeneFL arrays that contained probes representing 6800 full-length human genes. The Student t test and Mann-Whitney ranking were used independently to select candidates with probability values <.05. Hierarchical clustering analysis was performed on the selected candidates to assess the ability of these genes to discriminate between normal and affected individuals. RESULTS: Tissue inhibitor of metalloproteinases-1 and estrogen receptor-alpha messenger RNA expressions were found to be similar between uterosacral ligament and periurethral vaginal tissue in six participants. Of the 90 candidate genes that were identified, 62 genes were up-regulated and 28 were down-regulated in the stress urinary incontinence group. Genes that were involved in extracellular matrix activity in the up-regulated group include transforming growth factor-beta3, laminin, and collagen type VI. Down-regulated genes that may participate in collagen metabolism include laminin-related protein, collagen XVII, serine/threonine protein kinase, type II interleukin-1 receptor, and platelet-derived growth factor-associated protein. CONCLUSION: In this preliminary study, we identified differential gene expressions that may contribute to extracellular matrix remodeling in pelvic tissue from women with stress urinary incontinence in the proliferative phase versus continent control subjects. The alteration in expression of these candidate genes suggests that they should be targets for further investigation.

Adult↗
PubMed2001

RNAi-mediated oncogene silencing confers resistance to crown gall tumorigenesis.

Crown gall disease, caused by the soil bacterium Agrobacterium tumefaciens, results in significant economic losses in perennial crops worldwide. A. tumefaciens is one of the few organisms with a well characterized horizontal gene transfer system, possessing a suite of oncogenes that, when integrated into the plant genome, orchestrate de novo auxin and cytokinin biosynthesis to generate tumors. Specifically, the iaaM and ipt oncogenes, which show approximately 90% DNA sequence identity across studied A. tumefaciens strains, are required for tumor formation. By expressing two self-complementary RNA constructions designed to initiate RNA interference (RNAi) of iaaM and ipt, we generated transgenic Arabidopsis thaliana and Lycopersicon esculentum plants that are highly resistant to crown gall disease development. In in vitro root inoculation bioassays with two biovar I strains of A. tumefaciens, transgenic Arabidopsis lines averaged 0.0-1.5% tumorigenesis, whereas wild-type controls averaged 97.5% tumorigenesis. Similarly, several transformed tomato lines that were challenged by stem inoculation with three biovar I strains, one biovar II strain, and one biovar III strain of A. tumefaciens displayed between 0.0% and 24.2% tumorigenesis, whereas controls averaged 100% tumorigenesis. This mechanism of resistance, which is based on mRNA sequence homology rather than the highly specific receptor-ligand binding interactions characteristic of traditional plant resistance genes, should be highly durable. If successful and durable under field conditions, RNAi-mediated oncogene silencing may find broad applicability in the improvement of tree crop and ornamental rootstocks.

Arabidopsis↗
PubMed1970

Chromosomal distribution of rapidly reannealing DNA in Drosophila melanogaster.

Cytological hybridization has been used to localize fractions of rapidly reannealing DNA in salivary chromosomes of Drosophila melanogaster. Complementary RNA of high specific activity was transcribed from hydroxyapatite-fractionated rapidly reannealing sequences and from selected buoyant-density fractions of total DNA. It was then hybridized to chromosome squashes after denaturation of DNA in NaOH. Highly "repeated" DNA sequences were detected over much of the chromosome, but were concentrated in chromocentric heterochromatin. A family of sequences with a low percentage of guanosine plus cytidine was highly concentrated in a particular region within the chromo-center. One "euchromatic" region near the tip of chromosome arm 3L also exhibited a concentration of repeated sequences.

Animals↗
PubMed1972

Assignment of three human genes to chromosomes (LDH-A to 11, TK to 17, and IDH to 20) and evidence for translocation between human and mouse chromosomes in somatic cell hybrids (thymidine kinase-lactate dehydrogenase A-isocitrate dehydrogenase-C-11, E-17, and F-20 chromosomes).

Independently derived man-mouse somatic cell hybrids and their derivative subclones show a positive correlation between the expression of human lactate dehydrogenase A subunits and the occurrence of the human C-11 chromosome. Data are also presented that confirm the previously reported linkage of the thymidine kinase locus to the E-17 chromosome. A translocation of the E-17 chromosome provides presumptive evidence for the assignment of the thymidine kinase locus to the long arm segment of the E-17 chromosome. This translocation also provides evidence for translocation between man and mouse chromosomes in somatic cell hybrids. A presumptive association between the human phenotype for isocitrate dehydrogenase and the human F group is also described. Identification of specific human chromosomes was achieved by the application of several new cytological techniques: measurement of chromosome arm length, in situ annealing with mouse satellite complementary RNA, constitutive heterochromatin staining with Giemsa, and quinacrine mustard fluorochromatic staining.

Animals↗
PubMed1972

Widespread presence, in chickens, of DNA complementary to the RNA genome of avian leukosis viruses.

DNA-RNA hybridization experiments have demonstrated the widespread presence in chickens of DNA complementary to the RNA of avian myeloblastosis virus. All apparently normal chicken embryos, or adult chickens that were tested, contained viral DNA in amounts ranging from 1.7 to 4.6 viral genome equivalents per cell. Embryos that were negative or positive for the group-specific antigen of avian leukosis viruses contained the same amount of viral DNA. Embryos from a strain of chickens free of leukosis viruses of groups A and B that develop 40% fewer spontaneous leukotic tumors than the original strain contained an average of 2.1 viral DNA equivalents per cell, whereas the original strain contained 3.2 viral DNA equivalents. By comparison, leukemic cells and cells infected with avian myeloblastosis virus or Rous sarcoma virus contained between 4 and 13 viral DNA equivalents. This study provides direct biochemical evidence for the presence in normal chicken cells of avian leukosis virus DNA that is inheritable and that represents either complete or incomplete viral genomes.

Animals↗
PubMed1973

DNA of Epstein-Barr virus detected in tissue of Burkitt's lymphoma and nasopharyngeal carcinoma.

The association of Epstein-Barr virus (EBV) DNA with African tumors was examined by the method of hybridization with complementary RNA. 22 out of 23 biopsies of Burkitt's lymphoma and 18 out of 23 biopsies of nasopharyngeal carcinoma were found to contain EBV DNA. The number of EBV genome equivalents per cell ranged from four to 113. Five out of 24 other African tumors, two adenocarcinomas, and one each of melanoma, reticulum cell sarcoma, and carcinoma of the antrum, also contained EBV DNA.

Adenocarcinoma↗
PubMed1974

Direct evidence for the presence of Epstein-Barr virus DNA and nuclear antigen in malignant epithelial cells from patients with poorly differentiated carcinoma of the nasopharynx.

A well-differentiated squamous cell carcinoma and three poorly differentiated carcinomas of the nasopharynx were analyzed for the presence of Epstein-Barr virus DNA by hybridization with radioactive complementary RNA. The well-differentiated carcinoma contained no detectable Epstein-Barr virus DNA, whereas the three anaplastic carcinomas contained 41, 16, and 14 viral genome equivalents per cell. The anaplastic carcinomas were heavily infiltrated with lymphocytes and other non-neoplastic cells. All four tumors were successfully passaged in nude (thymusless) mice. Mouse cell admixture in the heterotransplanted tumors was estimated by analysis of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and varied between 25% and 80%. After two passages in nude mice, the carcinoma that was negative for Epstein-Barr virus DNA remained negative, while the three anaplastic carcinomas retained their viral DNA. After correction for mouse cell admixture, the latter tumors were found to contain about 80, 55, and 160 Epstein-Barr virus genome equivalents per human cell. The virus-determined nuclear antigen was localized in the large carcinoma cell clusters in all three mouse-passaged tumors positive for the viral DNA, but other virus-determined antigens were not detected, indicating the absence of virus induction. In contrast to the original tumor biopsies, the nude-mouse-passaged tumors showed virtually no lymphocytic infiltration. It is concluded that the Epstein-Barr virus DNA found in biopsies of human nasopharyngeal carcinomas is localized in the neoplastic cells.

Animals↗
PubMed1974

Replication of the resident repressed Epstein-Barr virus genome during the early S phase (S-1 period) of nonproducer Raji cells.

Replication of the resident repressed Epstein-Barr virus genome in synchronized nonproducer Raji cells was shown to occur during the early S phase (S-1 period) by hybridization of cell DNA with virus-specific complementary RNA (cRNA). The S-1 period was previously identified as the critical period for virus activation induced by thymidine analogues. The findings reported here and elsewhere are consistent with the proposal that: (i) virus activation is initiated at or near the site of association of the resident viral genome with cell DNA, (ii) replication of the resident virus genome in nonactivated cells is under cell control mechanisms, and (iii) the resident virus genome is physically associated with early replicating cell DNA.

Burkitt Lymphoma↗
PubMed1975

Localization of cytoplasmic-membrane-associated DNA in human chromosomes.

In situ hybridization was used to localize the sites of the repeated sequences of cytoplasmic-membrane-associated DNA (cmDNA) in human chromosomes. cmDNA was mainly present in the heterochromatic regions of chromosomes 9. Although cmDNA has buoyant densities in neutral and alkaline CsCl gradients similar to those of human satellite C DNA, which also is localized in the heterochromatic region of chromosomes 9, cmDNA was not homologous to satellite C DNA as determined by DNA-complementary RNA hybridization. These results are in agreement with our previous studies, which indicated that cmDNA is a unique species of human DNA.

Binding Sites↗
PubMed1975

Epstein-Barr virus genomes with properties of circular DNA molecules in carrier cells.

A high-density fraction of high-molecular-weight DNA was isolated from the human lymphoid cell line Raji. This cell line contains 50 to 60 virus genome equivalents of Epstein-Barr virus DNA per cell, and the high-density DNA fraction was 10-fold enriched in such viral sequences. Sedimentation analysis on neutral glycerol gradients, followed by hybridization experiments with viral complementary RNA, showed that most of the intracellular viral DNA sequences in this material did not cosediment with the cellular DNA, but were recovered as two distinct species with sedimentation coefficients of 100 S and 65 S. These two forms sediment 1.70-1.75 and 1.10-1.12 times as fast as the linear Epstein-Barr virus DNA from virus particles, and thus have the hydrodynamic properties of a covalently closed circular form and a nicked (containing single-strand breaks) circular form of the virus genome. The 100S form also behaved as a covalently closed circular EBV DNA molecule on gradient centrifugation in CsC1/propidium diiodide, It would appear that latent Epstein-Barr virus DNA has the properties of a mammalian episome, and that both nonintegrated and integrated viral DNA sequences can be isolated from carrier cells.

Base Sequence↗
PubMed1976

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

The DNAs from five independent simian virus 40 (SV40) transformants of the BALB/c3T3 mouse cell line were digested with either the HpaII or the BamHI restriction endonuclease and the resulting fragments were fractionated by gel electrophoresis. The DNA fragments were denatured in situ in the gel and transferred to a membrane filter. Fragments containing viral DNA were detected by hybridization with high specific activity 32P-labeled SV40 complementary RNA (cRNA) synthesized in vitro. Each of the lines yielded a small number of fragments containing SV40 DNA and the fragments from each line were different. This observation shows that the structure of the integrated SV40 DNA and/or its location in the host DNA are different in each line.

Cell Line↗
PubMed1979

Sites of sequence variability in Epstein-Barr virus DNA from different sources.

The intracellular Epstein-Barr virus (EBV) DNA present in virus-transformed cells was partly purified from 23 cell lines or biopsies of Burkitt lymphoma, nasopharyngeal carcinoma, infectious mononucleosis, or healthy carrier origin. Such DNA was cleaved in fragments (A-K) of molecular weights between 1 x 10(6) and 30 x 10(6) with restriction enzyme EcoRI, and these fragments were analyzed by standard methods involving agarose gel electrophoresis, transfer to nitrocellulose filters, and hybridization with radioactive EBV DNA or complementary RNA. Sequence variability among different EBV DNA isolates was largely confined to the A, C, and I fragments. These results are discussed in relation to the linkage map of the EcoRI fragments of EBV DNA. The EcoRI cleavage pattern of intracellular viral DNA of an EBV-like virus from baboon cells, Herpesvirus papio, was entirely different from that of human EBV isolates.

Animals↗
PubMed1982

Properties of a cell-free system for synthesis of citrus exocortis viroid.

Partially purified nuclei from citrus exocortis viroid (CEV)-infected Gynura aurantiaca are able to synthesize linear and circular viroid molecules. Pretreatment of the nuclei with actinomycin D or deoxyribonuclease did not affect viroid synthesis, whereas the synthesis of other cellular RNAs was severely reduced. These observations support the essential role of CEV complementary RNA sequences in viroid replication. However, when alpha-amanitin was included in this in vitro synthesis system, CEV replication was markedly reduced by concentrations of 10 nM or greater. Taken together, these data support the proposition that viroid synthesis is catalyzed by a DNA-dependent RNA polymerase acting on a RNA template.

Journal Article↗