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PubMed1983

Partial amino terminal sequence of the precursor of mitochondrial ATPase inhibitor protein synthesized with mRNA partially purified by gel permeation chromatography.

Messenger RNA coding mitochondrial ATPase inhibitor protein, a small peptide comprised of 63 amino acid residues, was separated from a large quantity of mRNAs of larger molecules by high speed gel permeation chromatography. Messenger RNA coding a small stabilizing factor of inactivated F1F0-ATPase complex, which is also comprised of 63 amino acids, was recovered in the same fraction as the ATPase inhibitor, whereas mRNA for a large stabilizing factor with an apparent molecular weight of 15,000 was recovered in a fraction of slightly larger molecules. ATPase inhibitor precursor labeled with various kinds of radioactive amino acids was prepared separately by cell-free translation with the purified mRNA, and the amino terminal sequence of the precursor was examined. It was demonstrated that an extra peptide of 21 amino acid residues, including 5 leucine, 4 serine, 1 glycine, and 1 methionine residues, is located at the amino terminus of the ATPase inhibitor precursor.

Amino Acid Sequence↗
PubMed1981

The effect of 5-fluorocytosine on the synthesis of 80S ribosomes by pathogenic fungi.

We studied the influence of 5-fluorocytosine(5FC) on the monomeric 80S ribosomes isolated by gradient ultracentrifugation from Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus and Wangiella dermatitidis. Labelling of the cultures with 32PO4 and [3H]-leucine showed marked inhibition of both ribosomal RNA and ribosomal protein synthesized during the contact of the fungi with the drug. The degree of inhibition was very similar for the synthesis of both ribosomal constituents, (mostly 60-80% each during the initial hours of the contact). This indicates that some ribosomes were still synthesized in the presence of 5FC and that the drug inhibits the synthesis of complete ribosomes to the same extent as the synthesis of ribosomal RNA and protein. By labelling fungi with [14C]-5FC it was shown that in those 80S ribosomes that were still synthesized, large quantities of the drug were incorporated--doubtless as 5-fluorouracil (5FU) in RNA. It can be concluded that RNA containing 5FU is still capable of combining with protein to form complete ribosomes of normal conformation. This is, however, no proof that these ribosomes would have a normal function.

Cell-Free System↗
PubMed2001

Extraction of total RNA from adipocytes.

RNA isolation from adipocytes presents with several technical problems and yields unacceptable results when following standard protocols. Here, we will describe additional steps and modifications necessary for the use of different RNA isolation protocols in terms of RNA yield, RNA quality and preparation time. Using five times the recommended quantity of lysis buffer, incubating the lysate at 37 degrees C, repeatedly passing the lysate through a cannula, and centrifugation to remove the lipid layer are essential additional steps when working with adipocytes. With these modifications, isolation of total RNA resulted in an average yield of 12-30 microg total RNA from 2 x 10(6) cells. Preparation times were similar for all but the CsCl gradient method. The purest RNA was obtained by spin-column purification, whereas acid phenol-chloroform methods yielded the highest amounts of total RNA. CsCl gradient ultracentrifugation is suggested for situations where DNase I digestion is impractical.

Adipocytes↗
PubMed1989

Regulated expression of early and late RNAs and proteins from the human cytomegalovirus immediate-early gene region.

Expression of RNA and protein from the human cytomegalovirus immediate-early (IE) gene region (map units 0.732 to 0.751) was analyzed at early and late times after infection. The level of RNA present at late times (48 to 72 h after infection) was significantly higher than that present at IE times (5 h after infection). The profile of IE RNA in the cytoplasm of infected cells was different from that previously reported on polysomes (R. M. Stenberg, P. R. Witte, and M. F. Stinski, J. Virol. 56:665-675, 1985). The data indicate that the 1.95-kilobase (kb) major IE region 1 mRNA, which codes for the 72-kilodalton (kDa) protein, and the 1.7-kb IE region 2 (IE2) spliced mRNA, which codes for the IE2 55-kDa protein, may be preferentially associated with polysomes. However, the IE2 2.2-kb unspliced mRNA, which codes for an 86-kDa protein, may be preferentially excluded. This RNA was abundant in the cytoplasm under IE conditions but was not present on polysomes in significant quantities. This indicates that IE gene products may be involved in translational control of cytomegalovirus RNA. At late times, new transcription takes place within region 2. A 1.5-kb RNA is transcribed from a late promoter in region 2 that apparently does not function in cells infected with DNA-negative mutant ts66. These results demonstrate that the IE gene region is transcribed throughout infection and that multiple levels of regulation exist.

Antigens, Viral↗
PubMed1998

Gigantism in a bacterium, Epulopiscium fishelsoni, correlates with complex patterns in arrangement, quantity, and segregation of DNA.

Epulopiscium fishelsoni, gut symbiont of the brown surgeonfish (Acanthurus nigrofuscus) in the Red Sea, attains a larger size than any other eubacterium, varies 10- to 20-fold in length (and >2, 000-fold in volume), and undergoes a complex daily life cycle. In early morning, nucleoids contain highly condensed DNA in elongate, chromosome-like structures which are physically separated from the general cytoplasm. Cell division involves production of two (rarely three) nucleoids within a cell, deposition of cell walls around expanded nucleoids, and emergence of daughter cells from the parent cell. Fluorescence measurements of DNA, RNA, and other cell components indicate the following. DNA quantity is proportional to cell volume over cell lengths of approximately 30 micrometers to >500 micrometers. For cells of a given size, nucleoids of cells with two nucleoids (binucleoid) contain approximately equal amounts of DNA. And each nucleoid of a binucleoid cell contains one-half the DNA of the single nucleoid in a uninucleoid cell of the same size. The life cycle involves approximately equal subdivision of DNA among daughter cells, formation of apical caps of condensed DNA from previously decondensed and diffusely distributed DNA, and "pinching" of DNA near the middle of the cell in the absence of new wall formation. Mechanisms underlying these patterns remain unclear, but formation of daughter nucleoids and cells occurs both during diurnal periods of host feeding and bacterial cell growth and during nocturnal periods of host inactivity when mean bacterial cell size declines.

Animals↗
PubMed1996

Circadian rhythm and the per ACNGGN repeat in the mole rat, Spalax ehrenbergi.

Individual variability in circadian locomotor activity has recently discovered in the blind mole rat, Spalax ehrenbergi. An interesting association was found between different circadian types and two DNA fragments, 5.6 and 5.9 kb long, that contain the ACNGGN repeat sequence, homologous to a part of the period gene of Drosophila. Nine of 12 arrythmic animals showed the 5.6-kb band, while 13 of 17 circadian rhythmic animals had the 5.9-kb band. This repeat exists also in the brain RNA of the mole rat, apparently in higher quantities during the sleeping phase, suggesting that an unusual protein(s), composed of a poly-Thr-Gly segment, affects in circadian rhythm.

Animals↗
PubMed2000

[Water births and the exposure to HIV].

The risk of a work related HIV-exposure or infection to midwives, or other HCW, in attending waterbirths of (possible unrecognized) HIV-positive women is unknown. Our goal was the quantification of the loss of blood of the childbearing woman after a waterbirth into the waterpool, in taking pool-water-samples of 14 different waterbirths and measuring the hemoglobin in the plasma, and then correlating the mean found loss of blood and the mean quantity of poolwater with a hypothetical HIV-RNA viral load of 10(3)-10(6) copies per milliliters (ml) blood. All attended waterbirths were evaluated with a questionnaire regarding: parity of the child-bearing woman; serostatus for HIV/Hepatitis-B (HBV) and Hepatitis-C (HCV); length of the birth-process; perineum-rupture or not, etc. Questions concerning the HCW in implementing universal precautions like: type of gloves and garment used, if at all; duration of water contact with the hands; existing skin lesions; HBV-vaccination-status; years of professional experiences as a midwife; how many waterbirths attended etc. were also evaluated. The mean calculated loss of blood into the pool was 300 ml, the mean pool-water content 633 liters. With a hypothetical (maximal) HIV-RNA viral load of 10(6) copies per ml blood, we calculated a mean HIV-RNA viral load of 476 copies per ml pool-water. We also found 37% of the interviewed midwives (n = 14) to have skin lesions on hands or fingers; 1 received splashes into her (unprotected) face and 1 was not immunized against HBV. The mean loss of blood of 300 ml into the pool is a relevant amount. The skin-contact of the HCW with the potentially contaminated water is the norm, because of the failure of the type of used gloves. Because of the diluting effect of the poolwater, we estimate the potential risk for a HIV-exposure to intact skin as minimal and, therefore a potential HIV-infection as "low level" and to be unlikely. However, a risk for nosocomial HBV-infection is significantly higher. We recommend wearing long-sleeved gloves, waterproofed garment, and HBV-vaccination to all HCW.

Baths↗
PubMed1984

RNA synthesis in pig follicular oocytes. Autoradiographic and cytochemical study.

RNA synthesis in pig oocytes was studied using autoradiography and silver staining of the nucleolus organizing region. Both methods confirmed that oocytes from the smallest follicles (0.5-0.7 mm in diam.) very intensely synthesize nuclear and nucleolar RNA. The nucleolar area of oocytes originating from follicles of 1.6-2.2 mm in diam. was labelled mainly on its periphery. After short pulse labelling (15 min) of oocytes from follicles of 5-6 mm in diam. only the nucleoplasm was labelled. The nucleolus had no significant labelling. The possibility that labelling of the compact nucleolus after a longer pulse represents migration of the newly synthesized nuclear RNA into the compact nucleolus, is discussed. The quantity of silver-positive material in dictyate oocytes significantly decreased as pig follicles enlarged in diam. from 2 mm to 5-6 mm.

Animals↗
PubMed1990

Correction of CD18-deficient lymphocytes by retrovirus-mediated gene transfer.

Leukocyte adhesion deficiency (LAD) is an inherited disorder of leukocyte function caused by derangements in CD18 expression. The genetic and functional abnormalities in a lymphocyte cell line from a patient with LAD have been corrected by retrovirus-mediated transduction of a functional CD18 gene. Lymphocytes from patients with LAD were exposed to CD18-expressing retrovirus and enriched for cells that express CD11a and CD18 (LFA-1) on the cell surface. Molecular and functional analyses of these cells revealed (i) one copy of proviral sequence per cell, (ii) viral-directed CD18 RNA that exceeded normal endogenous levels, (iii) normal quantities of CD11a and CD18 protein on the cell surface, and (iv) reconstitution of LFA-1-dependent adhesive function.

Animals↗
PubMed2006

Oxidized low-density lipoprotein-dependent endothelial arginase II activation contributes to impaired nitric oxide signaling.

Oxidized low-density lipoprotein (OxLDL) impairs NO signaling and endothelial function, and contributes to the pathogenesis of atherosclerosis. Arginase reciprocally regulates NO levels in endothelial cells by competing with NO synthase for the substrate l-arginine. In human aortic endothelial cells, OxLDL stimulation increased arginase enzyme activity in a time- and dose-dependent manner. Arginase activity reached its maximum as early as 5 minutes, was maintained for a period of more than 48 hours, and was associated with a reciprocal decrease in NO metabolite (NOx [nitrite and nitrate]) production. Furthermore, OxLDL induced arginase II mRNA expression after 4 hours. Small interfering RNA targeted to arginase II decreased both the quantity and the activity of arginase from baseline, prevented OxLDL-dependent increases in arginase activity, and induced an increase in NOx production. Immunofluorescence analysis revealed an association of arginase II with the microtubule cytoskeleton. Microtubule disruption with nocodazole caused a dramatic redistribution of arginase II to a diffuse cytosolic pattern, increased arginase activity, and decreased NOx production, which was restored in the presence of the specific arginase inhibitor (S)-(2-boronoethyl)-l-cysteine (BEC). On the other hand, epothilone B prevented microtubule disruption and inhibited OxLDL-dependent increases in arginase activity and attenuated OxLDL-dependent decreases in NOx. Preincubation of rat aortic rings with OxLDL resulted in an increase in arginase activity and a decrease in NOx production. This was reversed by arginase inhibition with the BEC. Thus, OxLDLs increase arginase activity by a sequence of regulatory events that involve early activation through decreased association with microtubules and a later increase in transcription. Furthermore, increased arginase activity contributes to OxLDL-dependent impairment of NOx production. Arginase, therefore, represents a novel target for therapy in atherosclerosis.

Animals↗
PubMedDate not supplied

A comparison of cell wall disruption techniques for the isolation of intracellular metabolites from Pleurotus and Lepista sp.

Different techniques were compared for their effectiveness in the disruption of the rigid cell walls of Basidiomycetes. Grinding under liquid nitrogen, stirred glass bead milling and enzymatic cell lysis were applied to the mycelia of Pleurotus sapidus and Lepista irina grown submerged. Each of the disruption procedures was evaluated by testing the quantity and quality of released intracellular metabolites: DNA, RNA, enzymes, and secondary metabolites. The most suitable method for nucleic acid isolation was grinding under liquid nitrogen, while bead mill homogenization was the superior technique for isolation of active enzymes. A new effective method is proposed for isolation of secondary metabolites with the aid of bead milling of fungal mycelia.

Agaricales↗
PubMed2000

Chronic lymphocytic leukemia B cells inhibit spontaneous Ig production by autologous bone marrow cells: role of CD95-CD95L interaction.

A variable degree of humoral immunodeficiency is a common feature in patients with B-cell chronic lymphocytic leukemia (B-CLL). The aim of this study was to explore the possibility that B-CLL cells play a direct role in this phenomenon. To this end, patients' bone marrow (BM) immunoglobulin (Ig)-secreting cells were cocultured with autologous purified B-CLL cells. The results show that tumoral cells inhibited the spontaneous IgG secretion by BM plasma cells, and this effect increased after PMA-induction of B-CLL cells. This inhibitory process was proportional to the number of B-CLL cells added and depended on cellular contact. Adhesion molecules did not appear to be involved in the cellular interaction, because the inclusion of blocking antibody to a variety of these proteins did not reverse the inhibitory phenomenon. However, the addition of monoclonal antibody that blocked the function of either CD95 or CD95L clearly reversed B-CLL cell inhibition on autologous BM plasma cells. These latter cells were shown to express CD95, and B-CLL cells contained detectable quantities of CD95L at the level of messenger RNA and protein. Annexin V-binding experiments revealed increased apoptosis of BM Ig-secreting cells when cocultured with autologous B-CLL cells. Finally, this inhibitory phenomenon might be operative in vivo because (a) there was a good correlation between the intensity of the inhibitory effect in vitro and the serum IgG level exhibited by every patient and (b) B-CLL cells also inhibited in vivo antigen-induced IgG-tetanus toxoid-secreting cells obtained from normal immunized subjects. Collectively, these data suggest that B-CLL cells inhibit autologous CD95-bearing Ig-secreting cells by the interaction with CD95L present on B-CLL cells and, hence, contribute to the state of humoral immunodeficiency that occurs in these patients.

Adult↗
PubMed2006

[Comparison of different methods for the detection of enteroviruses in drinking water].

UNLABELLED: Detection of enteroviruses in drinking water poses a very specific problem, since a very small number of particles have to be identified in huge water quantities. Currently, there are a number of methods to identify the concentration of virus particles and RNA templates to carry out RT-PCR, however, no standard method has yet been proposed. AIM: The aim of this report is to suggest optimal methods for the preparation of RNA templates to carry out RT-PCR. MATERIAL AND METHODS: In this experimental study, two different methods were employed on preparing RNA template. The concentration of virus particles in a large (10 L) and small (1 L) quantity of water was determined by use of the electropositive microporous virology filter (method 1 and method 2). Elution and flocculation of the virals particles were performed by organic extraction (method 1) and inorganic extraction (method 2). The sensitivity of the methods was assessed by testing the artificially contaminated water with 10(1) to 10(5) virus-particles using 1-L and 10-L containers of water. RESULTS: Method 1 detected 10(5) and 10(4) virus particles, method 2 10(5), 10(4) and 10(3) virus particles from 10 L and 1 L of water, respectively, yielding a statistically significant difference (p<0.01; chi2 = 6.061). Using two-step RT-PCR with nested PCR method, enteroviruses were detected in 42/100 (42%) samples of surface drinking water and in 83% of the same samples using RT-PCR without nested PCR.

Enterovirus↗
PubMedDate not supplied

[Preparation of extracellular ribonuclease form Actinomyces rimosus 994].

By sequential acid treatment, gel filtration and KM-cellulose sorption a 18--20-fold purified preparation of ribonuclease with a yield of 50--60% was obtained from the culture liquid filtrate of Actinomyces rimosus 994. The preparation had a high specific activity of 450,000--600,000 units/mg protein, contained 85--98% protein, insignificant amounts of carbohydrates and hydroxytetracycline, and no quantities of DNase, phosphomonoesterases, phosphodiesterase or proteases. In RNA degradation (preparation of the total yeast RNA of the Sigma Co.) optimal results were obtained at 50 degrees C and pH 7.0--7.2 in phosphate buffer and 7.6--8.0 IN Tris-HCl buffer. The preparation was stable at high temperatures (80--100 degrees) in the wide pH range and during storage in the lyophilized form and in buffer solutions. RNase effect was inhibited by zinc, copper, iron and cobalt cations and activated by beta-mercaptoethanol, citrate and EDTA. Protamine sulphate and urea in low concentrations (0.01% and 1--4 M, respectively) accelerated and in high concentrations (1% and 8 M, respectively) terminated the enzyme reaction. With respect to many properties RNase from Act. rimosus 994 was similar to extracellular RNases, produced by other actinomycetes and fungi.

Actinomyces↗
PubMed2003

A simple and cost-effective method for producing small interfering RNAs with high efficacy.

Small interfering RNAs (siRNAs) are powerful RNA interference (RNAi) reagents for directed post- transcriptional gene silencing. Exogenous siRNA is frequently used in RNAi studies. However, due to profound differences in the activity of siRNAs targeted to different regions of a gene, several reagents may have to be screened for optimal activity. This approach is expensive due to the cost of chemical synthesis of RNAs. We report a technically simple, quick and cost-effective method for the production of siRNAs that makes use of in vitro transcription and deoxyribozyme digestion of the transcripts to produce the desired sequence and length. The method allows for several siRNAs to be produced in parallel at much reduced costs. The siRNAs produced with this method were tested in MDA-MB-231 human breast cancer cells for efficacy against the type 1 insulin-like growth factor receptor (IGF1R) mRNA and they caused dose-dependent inhibition of IGF1R expression comparable to that induced by chemically synthesised siRNAs of the same sequence. This method is also useful for producing long RNA fragments of defined length and sequence that may be difficult to synthesise chemically, and also for producing large quantities of RNAs for applications including structural studies and the study of interactions between RNA and other molecules, such as proteins, other nucleic acids and drugs.

Cost-Benefit Analysis↗
PubMed1999

High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme.

An improved method is presented for the preparation of milligram quantities of homogenous-length RNAs suitable for nuclear magnetic resonance or X-ray crystallographic structural studies. Heterogeneous-length RNA transcripts are processed with a hammerhead ribozyme to yield homogenous-length products that are then readily purified by anion exchange high-performance liquid chromatography. This procedure eliminates the need for denaturing polyacrylamide gel electrophoresis, which is the most laborious step in the standard procedure for large-scale production of RNA by in vitro transcription. The hammerhead processing of the heterogeneous-length RNA transcripts also substantially improves the overall yield and purity of the desired RNA product.

Base Sequence↗
PubMedDate not supplied

Simultaneous quantitation of cytokine mRNAs in interleukin-1 beta stimulated U373 human astrocytoma cells by a polymerisation chain reaction method involving co-amplification with an internal multi-specific control.

The measurement of cytokine mRNA levels is of fundamental importance in the understanding of diverse pathological states. We present a simplification of a polymerase chain reaction-based technique which permits the simultaneous measurement of up to 20 cytokine mRNAs, together with those of several other cellular products, including beta 2-microglobulin and beta-actin. The technique makes use of internal standards bearing multiple PCR primer sites which are identical to those on the mRNAs to be assayed. Known quantities of the standards are added to the cellular RNA and the mixture is co-reverse transcribed and co-amplified. The simplifications described here are based on the fact that each pair of amplicons accumulates in a constant ratio even in the plateau phase of amplification. As a result, no preliminary experiments to determine the limits of the exponential phase of amplification are necessary; the same number of cycles may be chosen for all the mRNAs to be measured, whatever their level in the mixture might be; pipetting errors are avoided since all calculations are based upon the relative quantities of co-amplified material. Here we illustrate the method through a quantitative study of the expression of cytokine mRNAs in U373 human astrocytoma cells before and after stimulation with IL-1 beta. Quantitation was carried out either by incorporating radioactivity in the amplicons or by fluorescence measurements after propidium iodide staining. Only very low numbers of transcripts for IL-6, IL-8, CSF-1, MCP-1 and either Gro alpha or Gro beta were detectable in unstimulated cells. The levels of these cytokine mRNAs increased dramatically following IL-1 beta stimulation and, in addition, transcription of IL-1 beta, TNF alpha, GM-CSF, G-CSF, Gro gamma and MCP-1, some of which have not previously been detected in U373, was initiated in the stimulated cells. At the same time we found that transcripts for IL-2, IL-3, IL-4, IL-5, IFN gamma, huMlP1 alpha and huMlP1 beta were totally absent in this cell line. These results suggest a potentially important role for astrocytes in the local amplification of inflammatory responses in the brain.

Astrocytes↗
PubMed1989

The vitamin D-binding protein, alpha-fetoprotein, albumin multigene family: detection of transcripts in multiple tissues.

The serum vitamin D-binding protein (DBP), a member of a multigene family that includes alpha-fetoprotein (alpha FP) and albumin (ALB), is expressed at high levels in the adult liver. Although several studies have demonstrated the presence of DBP on the cell surface of immunocytes and cytotrophoblasts, evidence of extra-hepatic DBP synthesis remains inconclusive. Using Northern blot analysis of RNA from multiple rat tissues, the DBP transcripts were present in the expected high levels in the adult liver. In addition, DBP mRNA was found in the adult kidney, testis, abdominal fat, and 18-day fetal yolk sac. The concentrations of DBP mRNA in these tissues is 100-1000-fold less than in the liver. Polymerase chain reaction amplification (PCR) of DBP, alpha FP, and ALB cDNAs, reverse transcribed from a wide variety of rat tissue RNA samples, was performed to examine their respective tissue-specific patterns of gene transcripts. The tissues in which DBP gene transcripts were noted by Northern analysis were paralleled at 20 cycles of PCR with liver much greater than kidney, yolk sac, testis, and abdominal fat. Similarly at 20 cycles, alpha FP-amplified fragments were detected in the yolk sac much greater than brain, testes, uterus, liver, and placenta; and ALB-amplified fragments in liver much greater than testis, uterus, placenta, and yolk sac. Unexpectedly, at 32 cycles of PCR, DBP, alpha FP, and ALB mRNAs could be detected at very low levels in all tissues examined. To examine the nature of the DBP mRNA from these nonhepatic sources, a Sprague-Dawley kidney cDNA library was screened, and a single cDNA recombinant was identified. This clone contained a full length DBP cDNA with 14 nucleotide and 3 predicted amino acid differences from the Fischer liver DBP cDNA reported previously. We conclude that the kidney and liver DBP mRNAs are transcription products of the same DBP locus and that DBP, alpha FP, and ALB genes display different spectra of tissue distribution. The unexpected finding that all three spliced RNA transcripts could be detected in at least trace quantities in all tissues studied suggests a leaky level of transcription by this multigene family or their transcription at low levels by an unidentified stromal cell common to all organs studied.

Amino Acid Sequence↗