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PubMed1980

Oligo(U)- and poly(U)-containing RNA of wheat embryo.

A UMP-rich RNA fraction was separated from the bulk cellular RNA by affinity chromatography of wheat embryo total RNA. The obtained preparation was heterogeneous and contained polyribonucleotide chain segments which were resistant to RNAase T1 and consisted mainly of UMP residues (87 mol%). The UMP-rich segments were of various sizes, including large oligonucleotides and polynucleotides (up to approx. 150 nucleotides in length). The oligo(U)- and poly(U)-containing RNA fraction occurred in a low amount (approx. 1% of total RNA) both in dry and germinating wheat embryos. However, at the onset of germination, labelled precursors were preferentially incorporated into the UMP-rich RNA species. The early-synthesized RNA appeared and underwent a considerable degradation within the cell nuclei. It is assumed that both delayed maturation of structural gene transcripts and rapid transcription of regulatory gene units during initial germination stages contribute to the transient abundance of newly made UMP-rich RNA in the early wheat embryos.

Base Sequence↗

PubMed1975

[Characteristics of poly(A) rich RNA in human lymphocytes under normal conditions and in chronic lympholeukemia].

The presence of poly(A) containing RNA bound specifically to millipore filters, adsorbed on poly(U) cellulose and resistant to the pancreatic RNAs effect has been demonstrated in total RNA of human lymphocytes. These fractions show a DNA-like nucleotid content, a rapid turnover and they are characterized by sedimentation constants within the range of 9.5--15 S. On the average, 24.6 per cent of the applied initial radioactivity of total RNA is absorbed on membrane filters of normal lymphocytes, whereas from 13.3 to 23.3 per cent- in the poly(U) cellulose column. The study of lymphocytes total RNA in patients with chronic lympholeucosis has indicated a great heterogeneity in the content of poly(A) sequences rich RNA fractions.

Centrifugation, Density Gradient↗

PubMed2002

Beneficial effects of fenoldopam treatment on renal function in streptozotocin-induced diabetic rats.

We have studied the effect of chronic treatment with dopamine D1 receptor agonist fenoldopam (1 mg/kg, i.p. daily for 6 weeks) on renal function and metabolic parameters in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg). STZ produced severe hyperglycemia, hypoinsulinemia, hypercholesterolemia, hypertriglyceridemia, hypertension and bradycardia. Fenoldopam treatment significantly reduced fasting but not fed blood glucose levels and lowered the blood pressure in diabetic animals. Significant change was not observed in insulin, cholesterol, triglyceride levels. Diabetic animals showed increase in AUCglucose and decrease in AUCinsulin during oral glucose tolerance test. Fenoldopam treatment did not significantly change these values in diabetic animals. STZ produced increase in serum urea, creatinine and blood urea nitrogen. Diuresis and urinary sodium retention was observed in diabetic animals. Renal hypertrophy was observed as seen from increased kidney weight/body weight ratio and increased total RNA content as well as decreased total DNA content. Fenoldopam treatment significantly lowered serum urea, creatinine and blood urea nitrogen. Urinary sodium retention was significantly reduced and renal hypertrophy was prevented with fenoldopam treatment as seen from the improved kidney weight/body weight ratio. Fenoldopam treatment significantly prevented reduction in total DNA content and increase in total RNA content further substantiating reduced renal hypertrophy. Our data suggest that STZ induced diabetes is associated with renal dysfunctions and fenoldopam treatment could be beneficial in a condition where diabetes mellitus co-exists with hypertension and compromised renal function.

Animals↗

PubMed1991

New Zealand white rabbits immunized with RNA-complexed total histones develop an autoimmune-like response.

The antibody response of rabbits immunized with a total histone mixture containing randomly coiled H1/H5, H2A, H2B, H3 and H4 devoid of DNA was investigated in direct and competitive ELISA. The antisera were tested with isolated histones and chromatin and with a series of overlapping synthetic peptides covering the entire sequences of the four core histones and two peptides of H1. It was found that the New Zealand (NZ) white rabbits immunized with the total histone (TH) mixture complexed with RNA produced IgG antibodies reacting with histones and with a number of histone peptides but not with chromatin. The antisera also contained IgG antibodies which bound components that correspond to common target antigens in autoimmune diseases such as native dsDNA, peptides of Sm-D antigen, ubiquitin, branched peptides of ubiquitinated H2A and poly(ADP-ribose). By competition experiments, it was shown that these antibodies corresponded to non-crossreacting antibody populations. New Zealand rabbits immunized with TH in the absence of RNA or random outbred rabbits immunized with the RNA-complexed histone fraction produced antibodies reacting with histone, chromatin and very few histone peptides, while no activity with non-related antigens was observed. The pattern of reactivity of antisera raised in NZ rabbits with RNA-complexed TH was found to be very similar to that observed in sera of patients with systemic lupus erythematosus while, in contrast, the antibody response was very different in NZ or outbred rabbits immunized with various native nuclear particles and with individual histones. Altered nucleosome particles rather than native nucleosomes may represent the antigenic stimulus giving rise to autoantibodies.

Animals↗

PubMed1999

Gene expression of the renal endothelin system in renal transplant recipients on cyclosporine A based immunosuppression.

BACKGROUND: Immunosuppressive therapy based on cyclosporine A (CsA) is potentially nephrotoxic, and each dose of CsA is followed by a transient increase in plasma endothelin (ET)-1. The aim of this study was to investigate the effect of CsA based immunosuppressive therapy on renal gene expression of the ET(A) and ET(B) receptor subtypes and preproET-1 in human transplant needle biopsies. METHODS: Twelve living donor renal transplant recipients, median age 51.5 years (range 24-63 years) were included in the study. Immunosuppressive therapy consisted of CsA, azathioprine, and prednisolone. Baseline renal cortical needle biopsies from the living donor kidneys were obtained just before nephrectomy. Follow-up biopsies were obtained from the same transplanted kidneys after 2-6 weeks of immunosuppressive therapy. We used a quantitative, competitive reverse transcriptase polymerase chain reaction assay to measure renal ET(A) and ET(B) receptor subtype mRNAs as well as preproET-1 mRNA levels in each of the biopsies. RESULTS: The renal ET system was not significantly altered by CsA-based immunosuppressive therapy. Median ET(A) mRNA level was 185 (range 35-244) at baseline, and 120 (11-189) amol/microg total RNA after CsA based immunosuppressive therapy (P=0.11). ET(B) mRNA level was 506 (209-1411) at baseline, and 463 (267-1609) amol/microg total RNA at follow-up (P=0.44) and preproET-1 mRNA level was 160 (112-392) before and 221 (187-361) amol/microg total RNA after immunosuppressive therapy based on CsA (P=0.58). CONCLUSION: This study indicates that 2-6 weeks of CsA-based immunosuppression neither significantly influences renal gene expression of the ET(A) or ET(B) receptor subtypes nor preproET-1 in living donor renal transplant kidneys.

Adult↗

PubMed1982

Efficient extraction and translation of Plasmodium falciparum messenger RNA.

A convenient method was employed for the efficient extraction of total RNA from Plasmodium falciparum. By depleting the total RNA of tRNA, it was shown that P. falciparum or P. lophurae tRNAs markedly stimulate the translation of P. falciparum mRNA in the rabbit reticulocyte cell-free system. Analysis of the cell-free products revealed the presence of proteins well over 200 000 molecular weight, with the majority of polypeptides having high molecular weights. Thus, this system can now be used to isolate and characterize specific mRNA molecules from P. falciparum.

Animals↗

PubMed1991

Inhibition of 5-fluorouracil activation by oxipurinol in preparations of normal rat tissues and Jensen sarcoma: tumor-selective stimulation by inosine.

The effects of inosine on oxipurinol-induced inhibition of 5-fluorouracil (FUra) activation were investigated in a transplantable rat tumor and three normal rat tissues in vitro. FUra activation directed toward RNA was assessed in preparations of small intestine and Jensen sarcoma by determining the uptake of 14C-labeled FUra into purified total RNA of each tissue under identical conditions of incubation. Oxipurinol reduced the incorporation of FUra into intestinal RNA by as much as 93% but inhibited FUra uptake into tumor RNA by only about 45%. In the presence of sufficient oxipurinol to induce inhibition at approximately these levels, inosine failed to stimulate FUra uptake into intestinal RNA, whereas 0.1 mM inosine restored FUra uptake into Jensen sarcoma RNA to the level found in oxipurinol-free controls, and 0.5 mM inosine increased FUra uptake 29% above the controls. Both tissues had a nearly equivalent enzymatic capacity to cleave inosine by phosphorolysis. In view of what is currently known about the mechanism of action of oxipurinol and the metabolism of inosine, these results suggested that under our in vitro conditions rat small intestine was incapable of utilizing ribose-1-phosphate derived from inosine to stimulate the conversion of FUra to 5-fluorouridylate (FUMP), whereas Jensen sarcoma efficiently used this source of ribose-1-phosphate to increase the production of FUMP from FUra. In the absence of oxipurinol, inosine increased FUra uptake into total RNA of Jensen sarcoma by as much as 57%, compared to inosine-free controls, but markedly inhibited FUra uptake into total RNA of the small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice.

To define the mechanism of cyclosporine (CsA)-induced apoptosis, we investigated the expression of apoptosis-related genes in experimental chronic CsA nephrotoxicity. Mice on a low-salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30 mg/kg/day), and sacrificed at 1 and 4 weeks. Apoptosis was detected with deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain, and the expressions of apoptosis-related genes were evaluated by reverse transcription-polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54 +/- 41 vs. 3 +/- 3, p < 0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p < 0.01). The CsA group showed a significant increase in Fas-ligand mRNA (0.20 vs. 0.02 amol/microgram total RNA, p < 0.05) and Fas protein expression (146% vs. 95%, p < 0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05) and CPP32 mRNA (0.18 vs. 0.03 amol/microgram total RNA at 4 weeks, p < 0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 rho mol/microgram/h, p < 0.05) and CPP32 protease (15.6 vs. 2.7 rho mol/microgram/h, p < 0.05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl-2 protein increased significantly in the CsA group (5.3-fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas-ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis-related genes is associated with CsA-induced apoptotic cell death.

Animals↗

PubMed2000

Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens.

To establish a quantitative method for analysis of gene expressions in small areas of tissue after paraffin embedding, preliminary validation experiments with RT-PCR and Western blotting were performed using methacarn-fixed rodent tissues and a cultured PC12 cell line. A total RNA yield of 52 +/- 15 ng/mm2, sufficient for a quantitative RT-PCR of many genes, could be extracted from a deparaffinized 10-microm-thick rat-liver section by a simple, single-step extraction method. The low concentration of contaminating genomic DNA and the resolution of ribosomal RNAs in RNA gel proved the purity and integrity of the extracted RNA samples, allowing PCR amplification of a long mRNA sequence and mRNA species expressing low copy numbers. PCR amplification of mRNA-derived target gene fragments could be achieved by optimizing the amount of total RNA for reverse transcription and the number of subsequent PCR cycles for each gene. By this validation, organ- and sex-specific mRNA expression could be detected in methacarn-fixed paraffin-embedded tissues without additional DNase treatment of RNA samples. RT-PCR analysis could also be performed with total RNA extracted from deparaffinized tissue dissected with a laser capture microdissection system. In addition, extraction of protein yielded 4.9 +/- 2.1 microg/mm2 from a 10-microm-thick rat-liver section, allowing a quantitative expression analysis of protein by Western blotting. Thus, in addition to its advantages for immunohistochemistry, methacarn-fixed paraffin-embedded tissue has benefits for analysis of both RNAs and proteins in the cells of histologically defined areas.

Acetic Acid↗

PubMed1998

Electroneutral Na-coupled cotransporter expression in the kidney during variations of NaCl and water metabolism.

The purpose of the present study was to analyze the long-term regulation of renal bumetanide-sensitive Na+-K+-2Cl- cotransporter and thiazide-sensitive Na+-Cl- cotransporter gene expression during changes in NaCl and water metabolism. Male Wistar rats exposed to high or low NaCl intake, saline loading, dehydration, water loading, and furosemide administration during 7 days were studied. Control groups had access to regular food and tap water. Rats were kept in metabolic cages for 4 days before and during the experiment to determine daily urinary electrolyte excretion and osmolarity. At the end of the experiment, creatinine clearance and serum electrolyte levels were also measured. Kidneys were excised and macroscopically subdivided into cortex and outer and inner medulla. Total RNA was extracted from each individual cortex or outer medulla by use of the guanidine/cesium chloride method. The Na+-K+-2Cl- cotransporter expression in outer medulla total RNA was assessed by nonradioactive Northern blot analysis and the Na+-Cl- cotransporter expression in renal cortex total RNA was assessed by semiquantitative polymerase chain reaction. Experimental maneuvers were adequately tolerated, and all groups developed the appropriate renal response to each challenge. However, the level of expression of both cotransporters did not change in any model, except for a 2.8-fold increase in the Na+-Cl- cotransporter expression during dehydration. We conclude that nephron adaptation to 7-day modifications in NaCl and water metabolism does not include changes in the amount of electroneutral sodium-coupled cotransporter gene expression at the mRNA level.

Animals↗

PubMed1995

Determination of the poly(A) tail lengths of a single mRNA species in total hepatic RNA.

Poly(A) tail length is important in the stability and translation of mRNA. We describe procedures for the rapid and reproducible analysis of poly(A) tail length of a single mRNA species contained in a sample of total hepatic RNA. A short 3' fragment of a specific mRNA is prepared by RNase H digestion of the targeted mRNA region annealed to a short DNA oligonucleotide. The length of the poly(A) tail of the 3' fragment is then determined by running the sample on a polyacrylamide gel, by electrophoretic transfer, by probing with a radiolabeled cDNA and by comparing the size of the detected region with a specific RNA ladder or a DNA ladder.

Animals↗

PubMed1996

Detection and quantitation of perlecan mRNA levels in Alzheimer's disease and normal aged hippocampus by competitive reverse transcription-polymerase chain reaction.

Our previous studies have implicated perlecan, a specific heparan sulfate proteoglycan, in the pathogenesis of fibrillar beta-amyloid protein (A beta) accumulation and persistence in Alzheimer's disease (AD) brain. In the present investigation, we determined if perlecan mRNA was present in rodent and human brain tissue and whether perlecan persistence in A beta amyloid deposits in AD hippocampus may be partly due to increased perlecan expression and/or decreased perlecan degradation. To detect and to quantify low-abundance perlecan mRNA in rodent and postmortem human brain tissue, regions of perlecan domain I (503 and 366 bp) containing the unique heparan sulfate glycosaminoglycan attachment sites were analyzed by reverse transcription (RT) and polymerase chain reaction (PCR). Perlecan mRNA was detected in rodent brain, kidney, and liver and in human AD and normal aged frontal cortex. Different-size transcripts of perlecan domain I were found, suggesting the existence of alternatively spliced variants of perlecan or closely related gene products. Quantitative competitive RT-PCR using a mutant perlecan domain I internal standard was used to determine perlecan mRNA levels in total RNA isolated from the hippocampus of 10 AD (mean +/- SEM duration of illness, 11.3 +/- 1.4 years) and 10 normal aged controls. No significant difference in perlecan mRNA levels from the hippocampus of AD (1.12 +/- 0.29 amol/500 ng of total RNA) versus normal aged controls (1.09 +/- 0.30 amol/500 ng of total RNA) was found, indicating that perlecan expression remained at steady-state levels. These results therefore suggest that perlecan persistence in A beta-amyloid deposits in late-stage AD may be primarily due to decreased perlecan degradation and removal.

Aged↗

PubMed1994

Hepatic lipase gene is transcribed in rat adrenals into a truncated mRNA.

Rat adrenals contain a lipase activity that is indistinguishable from hepatic lipase (HL) present in liver. Expression of HL mRNA in adrenals was studied using the method of reverse transcription-polymerase chain reaction (RT-PCR). A 596-bp fragment of HL cDNA spanning exons 5 to 8 was amplified when using total RNA from rat adrenals and liver, but not from heart or kidney. The abundance of HL mRNA was quantified by competitive RT-PCR using a standard RNA that was generated in vitro by transcription from a deleted HL cDNA construct. Adrenals contained 0.4 attomoles of HL mRNA per microgram of total RNA, compared to 16 attomoles in liver. In hypertrophic adrenals isolated from corticotrophin-treated rats, the abundance also amounted to 0.4 attomoles of mRNA per microgram of total RNA. However, amplification of full-length cDNA from either control or hypertrophic adrenals was never observed. Detailed analysis by PCR using different combinations of primers indicated that exons 3 to 9 including the 3'-untranslated region were expressed in adrenal RNA, but not the first two coding exons. The upstream part of the adrenal lipase mRNA was cloned after rapid amplification of cDNA ends (RACE). The resulting clones showed a unique 126-bp sequence 5' of the exon 2-exon 3 junction. This sequence contained multiple termination codons in all three reading frames but lacked a potential start codon. RT-PCR using an HL-specific primer and an oligonucleotide directed against this 5'-sequence showed that it is not only expressed in RNA from adrenals but also in liver. Pulse-labeling of freshly isolated adrenocortical cells with [35S]methionine followed by immunoprecipitation with anti-HL antibodies failed to show synthesis of mature HL, but indicated the synthesis of immunoreactive proteins in the 40-45 kDa range that remained mainly intracellular. Hence, the HL gene is transcribed in adrenals but results in an mRNA species with a unique 5'-end. Translation from an internal start site may produce an intracellular HL isoform that differs markedly from the liver-type lipase previously identified in adrenals.

Adrenal Cortex↗

PubMed1983

[Effect of different doses of retinol acetate on the RNA and total protein content of rodent ovaries and endometrium].

The effects of different doses of retinol acetate (RA) on protein synthesis by the hamster ovaries and endometrium were studied. Two groups of CBA/C57BL mice aged 3-4 months were given 3.44% oily RA per os on a daily basis for 10 days. After completion of the experiments the paraffin sections 6 micron m thin were subjected to a histochemical assay for the content of RNA and total protein in the ovaries and endometrium. RA (50 000 IU) was found to stimulate RNA and protein synthesis in all the ovarian structures and endometrium. In the ovaries, vitamin primarily affected the follicular epithelium and internal the-ca of the follicles. Administration of RA in a dose of 80 000 IU inhibited protein synthesis in the ovaries and endometrium. As for the ovarian structures the greatest changes under toxic hypervitaminosis A (80 000 IU) were experienced by the follicular epithelium and ovocytes.

Animals↗

PubMed1969

Nucleic acid metabolism during cytokinin induced cellular differentiation.

Edstrom's microphoresis technique has been employed to determine the quantitative alterations in nucleic acid content and base composition of individual cells associated with the initiation of bud primordia in Funaria hygrometrica. The filamentous protonema of this moss initiates bud cells which through repeated divisions form the leafy gametophores. The cytokinin, 6-furfurylaminopurine (kinetin), was used to induce the differentiation of bud cells from protonematal cells. The total RNA content of kinetin-induced bud cells (22.0 mumug/cell) was nearly 15 times that of protonematal cells (1.6 mumug/cell). The same dramatic increase in total RNA was apparent in bud cells which developed spontaneously in older cultures. As would be predicted, the adenine (A) to guanine (G) ratio for DNA from bud and protonematal cells was identical (0.7). The A:G ratio for RNA from bud cells (1.0) was much lower than that from protonematal cells (1.7). Thus, kinetin-induced differentiation in this system involves a dramatic increase in total RNA, the base composition of which approaches that of DNA. The base composition (A:G ratio) of DNA remains constant.

Cell Differentiation↗

PubMed2004

Chronic administration of bumetanide upregulates calbindin D28k mRNA and protein abundance in rat distal convoluted tubules.

BACKGROUND/AIMS: Calbindin D28k has been reported to be involved in transcellular calcium transport along the rat distal convoluted tubule (DCT). It has also been shown that administration of bumetanide is associated with hypercalciuria. The experiments reported here were designed to verify whether chronic infusion of this diuretic affects the gene expression and protein abundance of calbindin D28k along rat kidney DCT. METHODS: Bumetanide was subcutaneously infused by an osmotic minipump for 7 days at a rate of 1.5 mg x h(-1) x kg(-1). cDNA was synthesized from total RNA extracted from DCT microdissected from collagenase-treated kidneys. RESULTS: Calbindin D28k mRNA abundance, quantified by competitive PCR, was found to be 13.7 +/- 1.9 amol x ng(-1) total RNA in DCT of control rats (n = 4) as compared to 24.2 +/- 2.4 amol x ng(-1) total RNA in DCT of bumetanide- treated rats (n = 5) (p < 0.01). This effect was associated with a 52% increase (p < 0.005) in calbindin D28k protein abundance, as detected by Western blot performed on tissue slices from renal cortex (n = 4). CONCLUSION: These data not only demonstrate that bumetanide upregulates the mRNA and protein abundance of calbindin D28k in rat DCT, but also suggest that DCT calcium reabsorption is increased following the administration of this loop diuretic.

Animals↗

PubMed2000

Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR.

The effects of serum on the expression of four commonly used housekeeping genes were examined in serum-stimulated fibroblasts in order to validate the internal control genes for a quantitative RT-PCR assay. NIH 3T3 fibroblasts transfected with an inducible chimeric gene were serum-starved for 24 h and then induced with 15% serum for 8 h. Serum did not alter the amount of total RNA that was expressed in the cells, however, the amount of mRNA significantly increased over time with serum-stimulation. Both messenger and total RNA from each of the time points were reverse transcribed under two different conditions; one in which the reactions were normalized to contain equal amounts of RNA and another series of reactions that were not normalized to RNA content. The resulting cDNA was amplified by real-time, quantitative PCR using gene-specific primers for beta-actin, beta-2 microglobulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18S ribosomal RNA. The expression of beta-actin and GAPDH increased up to nine- and three-fold, respectively, under all conditions of reverse transcription (P<0.01). The expression of 18S rRNA increased with serum-stimulation when the cDNA synthesized from non-normalized, total RNA was assayed (P<0. 01) but not when the reverse transcriptions were normalized to RNA content (P>0.05). The expression of beta-2 microglobulin increased up to two-fold when assayed from cDNA synthesized from non-normalized mRNA, but was unaffected by serum when the reverse transcriptions were normalized to mRNA. beta-2 Microglobulin expression was found to be directly proportional to the amount of mRNA that was present in non-normalized reverse transcription reactions. Thus, beta-2 microglobulin and 18S rRNA are suitable internal control genes in quantitative serum-stimulation studies, while beta-actin and GAPDH are not. The internal control gene needs to be properly validated when designing quantitative gene expression studies.

3T3 Cells↗

PubMed2006

Comparison of 'real-time' and immunometric RT-PCR: RNA interference of reverse transcriptase-PCR.

We measured interleukin-6 (IL-6) mRNA in whole blood, using an immunometric reverse transcriptase-polymerase chain reaction (IRT-PCR) method and 'real-time' RT-PCR using the Roche LightCycler. Both methods showed similar precision, in terms of inter- and intra-assay variation, where a lower total RNA concentration was used at a relatively high ratio of (IL-6) mRNA to total RNA. However, the 'real-time' RT-PCR method showed a greater loss of accuracy at higher concentrations of total RNA.

Humans↗