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At least 1,423 records · Page 79Linked to original sources
PubMed1991

Evidence for the presence of androgen receptors in human Leydig cells.

Localization of androgen receptors (ARs) in the human testis Leydig cells was examined with an AR assay and Northern blot analysis. Leydig cells, highly purified on a Percoll gradient, were used for the experiments. AR concentration in the total cell extract containing both the cytosol and nuclear fractions in Leydig cells was measured using [3H]methyltrienolone. ARs in Leydig cells showed a high affinity for [3H]methyltrienolone and the Kd and Bmax of the receptors were 1.24 nM and 11.7 fmol/mg protein, respectively. Northern blot analysis, using a 32P-labeled full-length human AR complementary DNA (cDNA) detected a 9.5-kb hybridizing band in the total RNA extracted from Leydig cells. These data can be interpreted as evidence of the existence of ARs in human Leydig cells.

Blotting, Northern↗
PubMed1984

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age.

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)+ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.

Aging↗
PubMed2003

Assessment of two methods for handling blood in collection tubes with RNA stabilizing agent for surveillance of gene expression profiles with high density microarrays.

Genome-wide expression studies of human blood samples in the context of epidemiologic surveillance are confronted by numerous challenges-one of the foremost being the capability to produce reliable detection of transcript levels. This led us to consider the Paxgene Blood RNA System, which consists of a stabilizing additive in an evacuated blood collection tube (PAX tube) and a sample processing kit (PAX kit). The PAX tube contains a solution that inhibits RNA degradation and gene induction as blood is drawn into the tube. The stability of RNA in PAX tubes under conditions for practical clinical applications has been determined by RT-PCR, but has not been assessed at the transcriptome level on Affymetrix microarrays. Here, we report a quality assured and controlled protocol that is capable of producing reliable gene expression profiles using the GeneChip system with RNA isolated from PAX tubes. Using this protocol, we compared quality metrics and gene-expression profiles of RNA, extracted from blood in PAX tubes that sat at room temperature for 2 h, with that of blood in PAX tubes incubated at room temperature for 9 h followed by storage at -20 degrees C for 6 days. Of numerous metrics, differences between the two handling methods were detected for the level of DNA contamination, RNA yield, and double stranded cDNA yield. Analysis of variance of gene-expression revealed small but significant differences between the handling methods. These results contribute to the determination of protocols for clinical studies and progress us towards the goal of using the transcriptome in diagnosis and surveillance.

Blood Specimen Collection↗
PubMed1974

Cell-free synthesis of adenovirus 2 proteins programmed by fractionated messenger RNA: a comparison of polypeptide products and messenger RNA lengths.

Cytoplasmic RNA extracted from human tissue culture cells infected with adenovirus type 2 was used to program protein synthesis in a cell-free system derived from mammalian cells. Analysis of the protein product by polyacrylamide gel electrophoresis revealed ten adenovirus-specific polypeptides. Five of these were further identified by analysis of tryptic peptides. Translation of RNA fractionated by sedimentation through sucrose gradients containing formamide demonstrated seven size classes of RNA, each of which programmed the synthesis of only one or two virus-specific polypeptides. Six of the virus-specific polypeptides were translated from RNAs much larger than expected for the size of the polypeptide.

Adenoviridae↗
PubMed1993

Nuclear angiotensin receptors induce transcription of renin and angiotensinogen mRNA.

The observation that nuclei from hepatic tissue exhibit specific angiotensin II (Ang II) binding led us to explore whether Ang II modulates mRNA in general, mRNA specific for renin system components, or both. Nuclei from hepatic tissue exhibited a single high-affinity (Kd = 0.4 nmol/L) Ang II-specific binding site, which was associated with increased RNA transcription. Whereas total RNA extracted from nuclei increased 1.5-fold in response to Ang II (10(-9) mol/L), specific mRNA for renin and angiotensinogen increased 7.8- and 2.5-fold, respectively. Ang II binding and induced transcription showed parallel Ang II dose responses that were both inhibited by 10(-5) mol/L DuP 753 or saralasin. Maximum Ang II binding and RNA transcription occurred at the same Ang II concentration (10(-9) mol/L). Higher doses of Ang II resulted in a progressive decrease in RNA transcription. Together, these results demonstrate that hepatic nuclei have functional Ang II-specific receptors. It is concluded that Ang II may elicit responses at nuclear receptors, which heretofore were associated only with Ang II receptors located on plasma membranes. However, the individual contribution of plasma and nuclear membrane Ang II receptors to the overall cellular Ang II transcriptional response and their possible interactions remain to be determined.

Angiotensin II↗
PubMed1989

Phenylephrine, vasopressin and angiotensin II as determinants of proto-oncogene and heat-shock protein gene expression in adult rat heart and aorta.

The expression of two oncogenes (conc) c-myc and c-fos, coding for nuclear proteins which play a regulatory role in growth and differentiation, and of two genes coding for two heat shock proteins (HSP) 68 (molecular weight 68,000) and 70 (molecular weight 70,000), which have a protective function during stress, have been investigated by Northern blot analysis of the total RNA, extracted from adult rat ventricle and aorta. (1) The two onc transcripts are absent from these tissues but their expression can be enhanced by a pretreatment with cycloheximide. (2) The HSP70 is, in part, constitutive, while HSP68 is not; both are thermo-inducible in an isolated coronary perfused rat heart. (3) The four messenger RNA (mRNA) are expressed in both ventricles and aorta, 1 or 2 hours after i.p. injection of 6 mg/kg phenylephrine or 12 IU/kg of vasopressin. (4) They are also induced by a continuous or discontinuous injection of angiotensin II (7.5 micrograms/kg per min) for 1-2 h, but only in the aorta. The lack of ventricular response to angiotensin II in rat ventricles has been attributed to the lack of angiotensin II receptors in this tissue. This indicates that, in addition to mechanical factors, circulating hormones which have in common the use of the phosphoinositol pathway, may activate the expression of genes coding for regulatory proteins. This may play a role in the genesis of both ventricular and aortic hypertrophy.

Angiotensin II↗
PubMedDate not supplied

[Detection of hepatitis A virus RNA by molecular hybridization].

Hepatitis A virus (HAV) was propagated in continuous rhesus monkey embryo kidney cells (FRhK-4 line). HAV RNA extracted with phenol from purified HAV pretreated with proteinase K was used for molecular cloning experiments. Two radioactive plasmids, pHAV-23 and pHAV-5p, containing HAV cDNA fragments complementary to structural and nonstructural parts of HAV genome, respectively, were found to be useful for discrimination between mature virions and subviral structures. HAV cDNA-RNA hybridization seems to be 10 times more sensitive than the enzyme immunoassay (ELISA).

Animals↗
PubMed1993

The developmental expression of three mammalian lens fiber cell membrane proteins.

PURPOSE: The expression of the lens fiber cell integral membrane proteins MP20, the alpha 3 gap junction protein (connexin 46), and MP26 (MIP) was determined both during development of the bovine fetal lens and in different animal species. METHODS: The relative levels of the mRNA species coding for these proteins was determined by Northern blotting; this was correlated with the amounts of these proteins as determined by Western immunoblotting. RESULTS: RNA extracted from fetal calf lenses at 3-4, 5-6, and 7-8 mo gestation, and from postnatal calf lenses, showed a progressive decrease of the mRNA species coding for MP20 (20-fold), alpha 3 gap junction protein (22-fold), and MP26 (12-fold) with development. This compared with a 6-fold decrease in total RNA and a 15-fold decrease in poly-A(+) RNA over the same period. In contrast to these significant changes in the levels of mRNA, there were no significant changes in the concentrations of these proteins over the same period. Whereas the mRNA coding for each of these proteins were concentrated in the lens cortical regions. MP20 and MP26 protein concentrations in cortical and nuclear regions were very similar. Both protein and mRNA coding for MP20, alpha 3 gap junction, and MP26 were detected in calf, sheep, pig, and rat lenses. CONCLUSIONS: These results indicate that the expression of the mRNA coding for the lens fiber cell integral membrane proteins MP20, alpha 3 gap junction, and MP26 is regulated and may be coordinated. That the decrease in mRNA concentrations is not correlated with changes in the concentrations of these lens membrane proteins indicates their unique stability.

Animals↗
PubMed1975

The submicroscopic structure of synthetically active units in a puff of drosophila hydei giant chromosomes.

Thin sections of nuclei of salivary glands of Drosophila hydei, treated in vitro with vitamin B-6 in the presence of either actinomycin D or 3'deoxyadenosine revealed the morphology of transcription units observed display a definite polarity with respect to the size of the RNP-particles attached to the deoxynucleoprotein matrix. The length of the longest unit observed was approximately 1.5 micronm, which corresponds well with the size of the RNA extracted from isolated puffs and with preliminary measurement of transcription units observed after speeading of the puffs according to the method of Miller and Beatty (1969).

Animals↗
PubMed1989

Turnip crinkle virus infection from RNA synthesized in vitro.

Genome-length cDNA clones of turnip crinkle virus (TCV) were constructed with SmaI and XbaI restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of modified lambda and T7 phage promoters such that in vitro transcription resulted in RNAs with 5 extra nucleotides at the 3' end, and 1, 2, or 14 extra nucleotides at the 5' end depending on the construction. Transcripts with 14 extraviral 5' nucleotides were not infectious, while transcripts with 1 or 2 additional 5' nucleotides, with or without 5'-cap analog included in transcription reactions, were biologically active. These were approximately an order of magnitude less infectious than RNA extracted from TCV virions. The additional 5' nucleotides were not maintained in progeny viral RNAs isolated from plants.

Cloning, Molecular↗
PubMed1985

In vitro study of the biological activity of RNAs after incubation of hog liver, heart and brain tissue at room temperature.

The biological activity of RNA, isolated from tissue which was incubated for 1, 3, or 6 hours at room temperature (simulation of post-mortem conditions), was preserved. However, the different organs used differ from each other. When liver is used, qualitative differences in the in vitro translation products are observed, after one hour incubation at room temperature, whereas when heart and brain are used these differences are not observed. We have also shown that relatively small amounts of post-mortem tissue is sufficient for RNA extraction. When using frozen tissue it is absolutely necessary to add RNase inhibitors during thawing to reduce the loss of biological activity.

Animals↗
PubMed1978

Requirement of protein synthesis for the degradation of host mRNA in Friend erythroleukemia cells infected wtih herpes simplex virus type 1.

We describe experiments which demonstrate that shortly after infection of Friend erythroleukemia cells with herpes simplex virus (HSV), polyribosomes dissociate and cellular mRNA degrades. Analysis of infected cell extracts on sucrose density gradients demonstrates that the majority of the polyribosomes have dissociated to monoribosomes at 2 h postinfection. Physical measurements of infected-cell RNAs support this conclusion and demonstrate that the polyadenylated RNAs decrease in size. The degradation of mRNA is apparently a stochastic process as judged by the failure to detect a shift in the Crt1/2 when polyadenylated RNA extracted from infected cells at different times is hybridized to globin complementary DNA. In experiments designed to determine whether dissociation of polyribosomes is sufficient to cause degradation of globin mRNA, the amount of globin mRNA in uninfected cells did not change when cells were treated with NaF or pactamycin at concentrations sufficient to dissociate all polyribosomes. In cells infected with UV-irradiated virus polyribosomes dissociate but globin mRNA does not degrade, suggesting that it is possible to separate dissociation from degradation.

Animals↗
PubMed1994

"Inapparent" hepatitis B virus infection and hepatitis C virus replication in alcoholic subjects with and without liver disease.

We evaluated hepatitis B virus DNA and hepatitis C virus RNA in sera from 110 HBsAg and IgM HBc antibody-negative heavy drinkers (50 cirrhosis, 13 chronic active hepatitis, 25 fatty liver with or without mild to moderate fibrosis, alcoholic hepatitis or both and 22 healthy alcoholic subjects) with polymerase chain reaction. Results of hepatitis C virus polymerase chain reaction were compared with those obtained with two tests (second generation recombinant immunoblot assay and enzyme-linked immunosorbent assay) used to detect hepatitis C virus antibodies. Hepatitis B virus DNA was found in three (2.7%) patients. Hepatitis C virus RNA was detected in 29 (29.8%) of the 97 subjects whose sera were well preserved for RNA extraction (42.5% cirrhosis, 83.3% chronic active hepatitis, 8% fatty liver and 0% healthy alcoholic subjects). Results obtained with second-generation recombinant immunoblot assay and enzyme-linked immunosorbent assay had a high degree of agreement with polymerase chain reaction as expected, the kappa indexes being 0.76 and 0.61, respectively. Nevertheless, five hepatitis C virus RNA-positive patients had negative recombinant immunoblot assay results, whereas all hepatitis C virus RNA-positive patients had positive or borderline enzyme-linked immunosorbent assay results. We conclude that, in Italian HBsAg-negative alcoholic patients, "inapparent" hepatitis B virus infection is rare. On the contrary, hepatitis C virus infection, as detected on hepatitis C virus polymerase chain reaction, is quite frequent, especially in patients who have cirrhosis and chronic active hepatitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Human coronavirus gene expression in the brains of multiple sclerosis patients.

Total RNA extracted from both white and gray matter of brain tissue from multiple sclerosis (MS) patients and controls was analyzed using a reverse transcription-polymerase chain reaction for the presence of the nucleic acid of human coronavirus (HCV) 229E and OC43, the two strains characterized to date and associated with respiratory infections. HCV-229E viral RNA was detectable in the central nervous system tissue of 4 of 11 MS patients and in none of 6 neurological and 5 normal controls. No HCV-OC43 nucleic acid was detected in any of the specimens. These results suggest a neurotropism on the part of the 229E strain of human coronavirus and underline the importance of further studies on its tissue distribution. The fact that it was detected only in tissue from MS patients illustrates the need for continued studies on the possible role of coronaviruses in the etiology of MS.

Base Sequence↗
PubMed2004

A sensitive and quantitative single-tube real-time reverse transcriptase-PCR for detection of enteroviral RNA.

BACKGROUND: Enteroviruses (EVs) are significant human pathogens. Rapid and sensitive diagnostic techniques are desirable. OBJECTIVES: To develop a quantitative single-tube real-time reverse transcription-polymerase chain reaction (RT-PCR) for human enterovirus ribonucleic acid (RNA) (QPCR), with protection against amplimer contamination. STUDY DESIGN: The method was evaluated with serial dilutions of EV, 62 cerebrospinal fluid (CSF) specimens from meningitis patients, and the third and fourth European Union Concerted Action Enterovirus Proficiency Panels. A commercial EV PCR test was run in parallel. RESULTS: Optimisation included RNA extraction procedure, design and concentrations of primers and probes from the 5' non-coding region as well as recombinant Thermus thermophilus polymerase (rTth), Mn(OAc)(2) and thermolabile UNG concentrations. Of 62 CSF samples from cases of meningitis submitted for QPCR testing, 34 (76%) and 21 (47%) were positive by QPCR and a commercial EV RNA detection kit, respectively. The detection limit of QPCR was 0.001 TCID(50)/ml (50% tissue culture-infective dose per millilitre) for a coxsackievirus B2 preparation and <10 copies of a plasmid containing coxsackievirus B2 complementary deoxyribonucleic acid (cDNA). The relation between threshold cycle (C(t)) and amount of virus was linear (r = 0.99) over a range of 10(-3) to 10(4) TCID(50)/ml of coxsackievirus B2. CONCLUSIONS: The QPCR method allows a large number of samples to be screened rapidly. Its sensitivity, simplicity, and reproducibility make it a suitable tool for the routine laboratory.

5' Untranslated Regions↗
PubMed1986

Characterization of cloned sequences complementary to F9 cell double-stranded RNA and their expression during differentiation.

In a cDNA library prepared from the RNA of cultured murine F9 teratocarcinoma cells, we identified sequences exhibiting strong hybridization to double-stranded RNA (dsRNA) of F9 cells but weak hybridization to mouse liver dsRNA. Northern-blot hybridization of RNA extracted from F9 cells with or without treatment with retinoic acid revealed differences in the expression of some of these sequences in undifferentiated and differentiated cells. As shown by Southern-blot hybridization experiments, these differences of expression were not related to a gross rearrangement of the corresponding genomic DNA sequences of the differentiated cells. When RNA from F9 cells was used, one of the cloned dsRNA-related sequences selected mRNA which was translated in vitro to a polypeptide with an Mr of 24,000; the level of this mRNA was reduced in F9 cells that had been treated with retinoic acid. Our results show that the differentiation of F9 cells induced by retinoic acid results in the differential expression of some middle-repetitive sequences.

Animals↗
PubMed1979

Experimental and clinical studies on toxicity of xenogeneic tumor-specific immune ribonucleic acid.

To learn the toxicity of xenogeneic tumor-specific immune ribonucleic acid (I-RNA), experimental and clinical studies were carried out. Experimentally, doses of 30 mg/kg, 15 mg/kg, 7.5 mg/kg or 3.75 mg/kg of xenogeneic I-RNA extracted from lymphoid tissues of rabbits sensitized with 105,000 Xg sediments of human gastric carcinoma tissue were injected intraperitoneally into Wistar rats once a day for 30 days. During the period of the study, changes of physiological and biochemical values were examined. In addition, pathological study was made for each organ after the I-RNA administration. There was no death during the study. In the groups of high dose administration, there were poor increase in the body weight, elevation of GOT, GPT and LDH, findings of vacuolar degeneration of hepatic cells, and increase of mesangial matrix and polynuclear glomerulus. Throughout the experiment, the groups given 7.5 mg/kg and 3.75 mg/kg of I-RNA showed no significant difference from control groups. Clinically, 31 cases treated with passive immunotherapy with allogeneic lymphocytes, preincubated with xenogeneic I-RNA, were examined retrospectively for the difference in hematological and biochemical data before and after the therapy. One case showed a transient mild febrile reaction after the therapy. There was no significant difference in hematological and biochemical data. Pathological findings in 3 autopsies after the treatment were reported.

Adult↗
PubMed1993

Presence of viral replicative intermediates in the liver and serum of patients infected with hepatitis C virus.

Hepatitis C virus (HCV) is a positive-polarity, single-stranded RNA virus, distantly related to the pestivirus and flavivirus genera. These viruses replicate through the formation of a minus-strand RNA intermediate, which encodes the positive-strand genome, which is subsequently encapsidated, enveloped, and released from infected cells. Minus-strand RNA is not found in the mature, circulating virions of flaviviruses. In an attempt to study the relative amounts of viral plus and minus strand in the liver and serum of HCV-infected individuals, we have developed a technique to amplify specifically each of the viral strands using a modified reverse transcriptase/polymerase chain reaction protocol on extracted RNA. Liver tumor and nontumor tissue from a patient with C-100-3 antibody was analyzed using this technique. In both cases, viral plus and minus strands were detected, although the plus-strand signal was several fold stronger than minus-strand signal by Southern hybridization. Sera from 11 C-100-3 antibody-positive patients with abnormal serum AIT levels were similarly analyzed. In all cases viral plus strand was detected, and in 10 of 11 cases viral minus strand was detected. The minus-strand signal was always much weaker than the plus-strand signal and the ratio of plus strand to minus strand varied among patients. No correlation was found between the level of minus strand detected or its ratio to plus strand with the level of serum transaminases or any other clinical parameter.

Adult↗