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At least 613 records · Page 34Linked to original sources
PubMed1998

[Antisense TGF-beta 1 transfection decreases acute and chronic rejection in allografts].

The process of chronic rejection (CR) threatens long-term organ graft survival and is the major remaining barrier preventing successful clinical transplantation. The etiology of CR remains speculative, but its correlation with acute rejection episodes, HLA mismatch and immunosuppressive non-compliance suggests that active immune attack is responsible. The authors hypothesize that transforming growth factor beta-1 (TGF beta-1) plays a causal role in regulating and modulating both the acute and the chronic rejection. To investigate this hypothesis in rats, recombinant adenoviruses (rAdv)-mediated gene transfer encoding downregulating antisense--or upregulating bioactive TGF beta-1 transgene were used to infect orthotopic aortic grafts. In a high responder MHC class I histocompatibility difference (ACI to Lewis rat) and in syngeneic controls (Lewis to Lewis) both expression vectors were detected 1, 2 and 12 weeks following transplantation by intragraft cytokine transcription. Aortic segments were divided and processed for histology and RNA extraction. TGF beta-1 RNA expression was then evaluated by semi-quantitative RT-PCR (s26 standardized, HPLC quantitated). Histological evaluation was performed by a transplant pathologist in a blinded fashion. All analysis were prospective and repeated in triplicate. Untreated allografts were used as background controls for the acute and chronic rejection showing an endogenous up-regulation of TGF beta-1 at early time points (1 wk 2.7 +/- 0.5; 2 wk 9.2 +/- 6.1) and a decreased TGF beta-1/s26 ratio in chronic rejection (12 wk 1.2 +/- 0.11). Successful rAdv-transgene activity was, however, detected in low levels in all aortic layers showing a 25%-35% transfection rate whereas beta-galactosidase control gene expression was found as far as 35 days post transplant. Administration of down-regulating antisense TGF beta-1 gene into transplant segments significantly decreased the intragraft TGF beta-1 transcription (1 wk 0.8 +/- 0.2; 2 wk 1.9 +/- 0.5; 12 wk 0.7 +/- 0.15) and was correlated with absence of ongoing acute graft rejection in allografts (p < 0.01) during the first 2 weeks. The degree of intimal hyperplasia proliferation was also decreased by 40% in chronic allograft rejection. The transfection of upregulating bioactive TGF beta-1 vector led to clear increase of the TGF beta 1 gene expression but had no significant effect on immune response either on syngeneic nor allogeneic grafts. These data suggest that TGF beta-1 plays a key role in modulating the early stage of acute rejection and is a crucial mediator of the outcome of chronic rejection. Down regulation of intragraft TGF beta-1 gene expression shows immunosuppresisve property and could be used to develop clinically relevant strategies in transplantation.

Adenoviridae↗
PubMed1988

Changes in expression of oncogenes and endogenous retroviral-like sequences during colon carcinogenesis.

The possible roles in experimental colon carcinogenesis of two protooncogenes (c-myc and c-H-ras), two endogenous retrovirus-related DNA sequences [rat leukemia virus (RaLV) and the 30S sequence], and two cell cycle related genes (beta-actin and ornithine decarboxylase) were studied by analyzing the levels of their corresponding RNAs during the course of azoxymethane induced and high fat promoted colon carcinogenesis. F-344 male rats received three s.c. injections of azoxymethane (15 mg/kg) or normal saline and were then subdivided into high or low fat diet groups. During subsequent serial sacrifices normal colon mucosa, adenomas, and carcinomas were harvested for histology and RNA extraction. Seventy-one RNA samples were analyzed by the Northern blot hybridization procedure using the appropriate 32P-labeled DNA probes. A marked increase in the abundance of c-myc, RaLV, and 30S RNAs were seen in all of the colon tumors, including adenomas and invasive carcinomas. No or a very low level of expression of RaLV and c-myc RNA was found in the flat grossly normal mucosa adjacent to the tumors and in the mucosa of the control rats. Some of the colon tumors also displayed increased levels of c-H-ras, ornithine decarboxylase and beta-actin RNAs but these findings were less striking and more variable than those seen with c-myc, RaLV, and 30S RNAs. These results suggest that increased expression of the c-myc protooncogene and of the endogenous retrovirus-like sequences (RaLV) and 30S are hallmarks of colon carcinogenesis in this model system.

Animals↗
PubMed1976

Effect of estrogen on gene expression in the chick oviduct. Effect of estrogen on the sequence and population complexity of chick oviduct poly(A)-containing RNA.

Total cellular RNA preparations were isolated from chicken oviducts at three different development stages: (a) immature chicks which were chronically stimulated with estrogen; (b) estrogen-stimulated chicks which were then withdrawn from hormone for 12 days; and (c) laying hens. Total cellular RNA containing 3'-poly(A) sequences (poly(A)-RNA) were than isolated from these preparations using oligo(dT)-cellulose chromatography. The number average nucleotide length of the poly(A)-RNA preparations in each case was approximately 2000 nucleotides. The number average nucleotide length of the poly(A) residues at the 3'-terminal end of each RNA preparation was approximately 70 adenylate residues. Complementary DNA (cDNA) copies to each preparation of poly(A)-RNA were synthesized using avian myeloblastosis virus RNA-directed DNA polymerase. The cDNApoly(A) preparations were then utilized in DNA excess hybridization experiments to analyze the complexity of the DNA sequences from which these RNAs were transcribed. Approximately 22% of each of the total cellular poly(A)-RNAs were transcribed from repeated DNA sequences (average repeat frequency of 35 copies/genome) while the remaining majority were transcribed from single copy or unique sequence DNA. It was possible to estimate the number of different poly(A)-RNA sequences per cell by analyzing the kinetics of hybridization of these cDNApoly(A) preparations to total cellular poly(A)-RNA extracts under conditions of RNA excess. The results revealed that 41% of the poly(A)-RNA from laying hen oviduct consisted of, on the average, three different sequences/cell, each of which was present in approximately 25,000 copies/cell. The remainder of the poly(A)-RNA in this tissue consisted of approximately 25,000 different sequences/cell, which were present largely in only two or three copies/cell. A somewhat similar sequence complexity was found for oviduct cells prepared from estrogen-stimulated chicks. We estimated that there were approximately 20,000 different poly(A)-RNA sequences/cell, each represented in only one to two copies/cell. However, there were five sequences which were present, on the average, in a concentration of 5600 copies/cell. The poly(A)-RNAs from hormone-wtihdrawn tissue, on the other hand, had a lower sequence complexity. There were only approximately 10,000 different poly(A)-RNA sequences/cell, each present in about three copies/cell. Furthermore, the few sequences present in a great abundance in hen and hormone-stimulated tissues were apparently absent in oviduct tissue from hormone-wtihdrawn chicks, suggesting that the intracellular concentrations of these high frequency RNA sequences are dependent on estrogen.

Animals↗
PubMed1981

Quantitative of murine mammary tumor virus-related RNA in mammary tissues of low- and high-mammary-tumor-incidence mouse strains.

Lactating mammary glands and hormonally induced mammary tumors of BALB/c mice from three geographically separated breeding colonies were examined by molecular hybridization, using murine mammary tumor virus (MuMTV) cDNA representing the entire viral genome to determine the amount of MuMTV-related RNA expressed in these tissues. The RNA extracted from these tissues by the classical sodium dodecyl sulfate-pronase, phenol-chloroform procedure (method 1) contained barely detectable levels of MuMTV-related sequences. In contrast, both normal lactating mammary glands and hormonally induced mammary tumors of these mice were found to contain approximately one to two copies of the MuMTV genome per cell by using a new procedure in which the RNA was extracted with guanidine derivatives (method 2). No significant differences in the MuMTV-related RNA content of the BALB/c mammary tissues were observed regardless of their colony of origin. Our results suggest that expression of MuMTV RNA does not change in BALB/c mammary glands during transformation to a malignant state and that MuMTV expression does not play a role in tumorigenesis in these mice. In view of the increased recovery of MuMTV-related RNA from BALB/c mice with method 2, we compared the level of MuMTV RNA expression in lactating mammary glands and mammary tumors of other mouse strains, including C57BL/6 and RIII, using both extraction methods. Yields of MuMTV-related RNA from mammary tissues increased by as much as 35- to 40-fold, using method 2 as compared with method 1. Therefore method 2, involving guanidine derivatives, appears to be method of choice for MuMTV-related RNA extraction from the mammary tissues of certain strains of mice, particularly those expressing relatively low levels of MuMTV RNA.

Animals↗
PubMed1976

Nuclear RNA in the mouse spleen at the peak of primary immune response.

Electrophoretic studies on nuclear RNA extracted from the spleens of immunized mice are reported. RNA was extracted by the phenol-detergent procedure from the purified nuclei of spleen cells at the peak of primary immune response after a single immunization with sheep red blood cells. The kinetics of labeling in vivo with 32P-orthophosphate indicate an accelerated biosynthesis of the ribosomal RNA precursors, 28S, 18S, 5S and 4S fractions of nuclear RNA in the spleens of immunized mice.

Animals↗
PubMed1966

Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid.

1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.

Animals↗
PubMed2006

An efficient method for purifying high quality RNA from wheat pistils.

Many methods are available for total RNA extraction from plants, except the floral organs like wheat pistils containing high levels of polysaccharides that bind/or co-precipitate with RNA. In this protocol, a simple and effective method for extracting total RNA from small and feathery wheat pistils has been developed. Lithium chloride (LiCl) and phenol:chloroform:isoamylalcohol (PCI) were employed and the samples were ground in microcentrifuge tube using plastic pestle. A jacket of liquid nitrogen and simplified procedures were applied to ensure thorough grinding of the pistils and to minimize the samples loss. These measures substantially increased the recovery of total RNA (approximately 50%) in the extraction process. Reliable differential display by cDNA-AFLP was successfully achieved with the total RNA after DNase treatment and reverse transcription. This method is also practicable for gene expression and gene regulation studies in floral parts of other plants.

DNA, Complementary↗
PubMed1994

Neuropeptide-Y gene expression in the goldfish brain: distribution and regulation by ovarian steroids.

Neuropeptide-Y (NPY) has been recently cloned from a goldfish cDNA library. Using a probe derived from this cDNA clone, the distribution and regulation of NPY mRNA in the goldfish brain were examined in the present study by in situ hybridization, Northern blot analysis, and ribonuclease protection assay. Frozen tissue sections from the goldfish brain and pituitary were hybridized with a digoxigenin-labeled RNA probe. Hybridization signal was detected mainly in fore-brain regions, particularly in the nucleus entopeduncularis of the ventral telencephalon, the preoptic area (POA), the olfactory bulbs, and various thalamic regions. In the midbrain, NPY mRNA was found in the optic-tectum and locus coeruleus. Northern blot hybridization of total RNA extracted from different brain areas with a 32P-labeled RNA probe detected a single mRNA species and confirmed that most NPY mRNA was present in telencephalon/POA and optic-tectum/thalamus. Our previous physiological studies showed that the ovarian steroids testosterone (T) and estradiol (E2) have potentiating effects on the actions of NPY on gonadotropin and GH release. Therefore, we also tested the possibility that T and E2 may modulate NPY gene expression. Fish were implanted ip with pellets containing T, E2, or no steroid (control) for 5 days, RNA was extracted, and NPY mRNA levels were estimated using a ribonuclease protection assay. Pretreatment with T or E2 induced a 2- to 3-fold increase in NPY mRNA levels in the telencephalon/POA, but not in the optic-tectum/thalamus. In situ hybridization using brains taken from T-implanted fish demonstrated that the site of steroid action is the POA. This report represents the first study on NPY gene expression in a nonmammalian species and demonstrates that 1) NPY mRNA is present in the neuroendocrine regulatory centers; and 2) ovarian steroids stimulate NPY gene expression in the POA.

Animals↗
PubMed1985

A circular dichroism study of the Turnip yellow mosaic virus-RNA.

We examined the circular dichroism spectra of intact Turnip yellow mosaic virus, freezed/thawed virus, empty capsid particles, and phenol extracted RNA. The circular dichroism signal of the empty capsid was found to contribute for less than 1% to the circular dichroism of the virus. Differences in the circular dichroism spectra indicate that TYMV-RNA exhibits different conformations when it is in situ in the virus, when it has been ejected by freezing/thawing and when it has been phenol extracted. Increase of the ionic strength up to 0.1 M NaCl led to conformational change of the RNA either freezed/thawed ejected or phenol extracted but not in situ in the capsid. Addition of spermidine (3 mM) induced a conformational change only for the phenol extracted RNA. These results are discussed with respect to the origin of the various conformational states of viral RNA.

Brassica↗
PubMed2003

Donor corneoscleral buttons: a new source of trabecular meshwork for research.

The human trabecular meshwork (TM) is the major site of resistance for aqueous humor outflow. The purpose of this investigation was to determine the suitability of TMs harvested from donor corneoscleral buttons (buttons) for laboratory studies. Histologic examination using light and electron microscopy was performed. Additionally, the effect of incubation in serum free media on histologic appearance was studied. Total RNA extraction was performed on 45 buttons and four whole eyes. Some TMs were used as a source for establishing primary cell cultures of TM endothelial cells. Compared with donor whole eyes, light microscopy showed comparable TM cellularity in the juxtacanalicular and corneoscleral TM; there was some loss of cells in the inner portion of uveal TM that was adjacent to the anterior chamber. The TM cells appeared healthy and structurally normal on transmissive electron microscopy. Usable quantities of total RNA could be extracted from buttons that had been stored up to 5 weeks. The amount of total RNA extracted correlated well with the histologic appearance. Incubation in serum free media did not have an effect on the histologic appearance. All attempts at establishing primary cell cultures were successful. Unused, donor corneoscleral buttons are an excellent source of TM.

Adult↗
PubMed1989

Glucocorticoid-receptor complexes are associated with small RNA in vitro.

Identification of RNA associated with soluble glucocorticoid-receptor complexes of HeLa cells was performed by immunoprecipitation of receptor complexes with a monoclonal antibody raised against rat liver glucocorticoid receptor. Polyacrylamide gel electrophoresis of RNA extracted from immunoprecipitates of cytosolic complexes revealed the presence of eight RNA bands, consisting of 28S, 18S, and small RNAs, including 5.8S, 5S and tRNA. A comparison of RNA species immunoprecipitated by monoclonal anti-glucocorticoid receptor antibody and IgG purified from normal mouse serum showed that four small RNAs were preferentially recovered after immunoprecipitation with anti-glucocorticoid receptor antibody. When these species were analyzed on sequencing gels, their nucleotide lengths coincided with those of 7-3, 7S, U2, and U1 RNA. Immunoprecipitation of nuclear extracts containing glucocorticoid-receptor-RNA complexes showed that the same set of small RNAs was preferentially immunoprecipitated by anti-glucocorticoid receptor antibody. The four small RNAs we detected represented minor species in whole extracts, and their preferential immunoprecipitation by anti-glucocorticoid receptor antibody was prevented by removal of glucocorticoid-receptor complexes from HeLa cell extracts. We conclude that 7-3, 7S, U2, and U1 RNA are associated with glucocorticoid-receptor complexes in vitro, and hypothesize that post-transcriptional effects of glucocorticoids may in part be mediated through interaction of receptor complexes with these small RNAs.

Antibodies, Monoclonal↗
PubMed2002

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.

Animals↗
PubMed2003

A rapid and effective method of extracting fully intact RNA from thermophilic geobacilli that is suitable for gene expression analysis.

Extraction of intact RNA is essential for quantitative gene expression analysis. Isolating high quality RNA from gram-positive bacteria is known to be problematic particularly from organisms that have optimal growth temperatures greater than 45 degrees C. We report a novel extraction protocol for the rapid isolation of fully intact RNA from thermophilic Geobacillus thermoleovorans using a lysing matrix containing a mixture of ceramic and glass beads, triisopropylnaphthalene sulfonic acid (TNS), and p-4-aminosalicyclic acid (PAS). Combining both detergents, TNS and PAS, appeared to increase denaturation of RNases at thermophilic temperatures. Gel electrophoresis revealed that only RNA isolated using the TNS-PAS procedure demonstrated sharp, undegraded 23S, 16S, and 5S ribosomal RNA bands. RNA extracted from geobacilli using commercially available kits was extensively degraded and was not suitable for detecting gene expression. Total RNA yields extracted with the TNS-PAS protocol were greater than eightfold higher than those obtained with available kits. Critically, it was also shown that only RNA isolated with the TNS-PAS-based method was suitable for monitoring thermophile gene expression patterns using RT-PCR analysis.

Aminosalicylic Acid↗