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At least 937 records · Page 52Linked to original sources
PubMed1989

Some properties of RNA from Physarum amoebae undergoing encystment: electrophoretic similarity to Acanthamoeba RNA.

Some properties of the RNA of myxamoebae-swarm cells of Physarum flavicomum undergoing microcyst formation, and during adenine inhibition of the developmental process, were compared. During the first 15 h incubation under encystment conditions, 11% more RNA was recovered from the adenine-inhibited cells (AIC) than from the normal control cells (NC), whereas the specific activity (SA) of the purified RNA from NC was 1.4 times higher than that from the AIC. Gel electrophoresis of purified total RNA revealed comigration of ribosomal RNAs (rRNA) of the myxamoebae with those of mouse kidney and liver, and diploid cells of P. flavicomum. In contrast to the apparently equivalent 18S and 28S pattern of the latter three RNAs, the quantity of the largest rRNA was significantly lower compared with that of the smaller (18S) rRNA in both NC and AIC myxamoebae-swarm cells. Total RNA from amoebae of a recent isolate of Acanthamoeba sp. quantitatively exhibited the same unusual pattern of rRNA distribution as the Physarum myxamoebae. A high pressure liquid chromatographic method was developed using a single solvent for the isocratic separation of the four major bases and seven methylated bases of RNA. Methylated adenine and guanine were detected in total RNA samples of myxamoebae of P. flavicomum. The mole % of uracil was slightly higher in the NC, whereas the mole % of adenine was slightly lower compared with that of the AIC. The guanine plus cytosine content of these purified total RNAs was about 61%.

Acanthamoeba↗
PubMed1993

Inhibition of mouse hepatitis virus multiplication by antisense oligonucleotide, antisense RNA, sense RNA and ribozyme.

Antisense nucleic acids against specific sequences of mouse hepatitis virus (MHV)-RNAs were tested for their inhibitory effects on viral multiplication in mouse DBT cells. An antisense oligonucleotide containing a sequence complementary to leader RNA was synthesized and shown to induce a significant inhibitory effect on the multiplication of MHV-JHM. A vector which expressed the antisense or sense mRNA7 of MHV was transfected into DBT cells. A decreased multiplication of MHV was observed in both cell lines. The transfected cell line which expressed ribozyme against the 5'-end of the MHV genome was established. The rate of inhibition of MHV-multiplication and the quantity of synthesized virus-specific mRNAs in this transfected cell line were the same for both antisense and sense RNA. These results show that antisense nucleic acids might be eligible for use as antiviral agents against MHV multiplication.

Animals↗
PubMed1991

Detection and identification of human influenza viruses by the polymerase chain reaction.

A series of oligonucleotide primers are described which hybridize to conserved regions of influenza virus cDNA and prime DNA synthesis in Taq polymerase catalyzed amplification reactions (PCR). Primers were designed to hybridize as nested pairs and, following a two-step amplification, produce uniquely sized DNA fragments diagnostic for viral type and subtype. Influenza A and B matrix-protein genes and the influenza C haemagglutinin gene were targets for the type-specific primers. Subtype-specific primers targeted conserved sequences within the three haemagglutinin or two neuraminidase subtypes of different human influenza isolates. The utility of this method was demonstrated using computer search methods and by accurately amplifying DNA from a variety of influenza A, B, and C strains. Type-specific primer sets showed a broad type specificity and amplified DNA from viral strains of unknown sequence. Restriction mapping and DNA sequencing showed that fragments amplified in this manner derived from the input template, confirming the accuracy of the method and demonstrating how PCR can be used to quickly derive sufficient sequence information for analysis of viral relatedness. Subtyping primers were able to distinguish accurately between the three haemagglutinin (H1, H2, H3) and two neuraminidase (N1, N2) alleles of human influenza A isolates. Again DNA was amplified from viruses of unknown sequence confirming that most of these primer sets may prove useful as broad range subtyping reagents. In order to simplify the work associated with analysis of many samples, we have also devised a rapid method for the isolation of viral RNA and synthesis of cDNA. Using this 'mini-prep' technique, it is possible to detect, amplify, and identify picogram quantities of influenza virus in a single day, confirming that PCR provides a useful alternative to existing methods of influenza detection.

Animals↗
PubMed1998

Cell-free human immunodeficiency virus type 1 in breast milk.

Breast-feeding may be an important route of human immunodeficiency virus type 1 (HIV-1) vertical transmission in settings where it is routinely practiced. To define the prevalence and quantity of HIV-1 in cell-free breast milk, samples from HIV-1-seropositive women were analyzed by quantitative competitive reverse transcription-polymerase chain reaction (QC-RT-PCR). HIV-1 RNA was detected in 29 (39%) of 75 specimens tested. Of these 29 specimens, 16 (55%) had levels that were near the detection limit of the assay (240 copies/mL), while 6 (21%) had >900 copies/mL. The maximum concentration of HIV-1 RNA detected was 8100 copies/mL. The prevalence of cell-free HIV-1 was higher in mature milk (47%) than in colostrum (27%, P = 0.1). Because mature milk is consumed in large quantities, these data suggest that cell-free HIV-1 in breast milk may contribute to vertical transmission of HIV-1.

Adolescent↗
PubMed1995

Regulation of coronavirus mRNA transcription.

Coronaviruses synthesize a nested set of six to eight subgenomic (sg) mRNAs in infected cells. These mRNAs are produced in different, but constant, molar ratios. It is unclear which factors control the different levels of sg mRNAs. To determine whether the intergenic sequence (IS) involved in sg mRNA synthesis could affect the transcription efficiencies of other ISs and in this way regulate transcription levels, we inserted multiple ISs at different positions into a mouse hepatitis virus defective interfering RNA. Quantitation of the sg RNAs produced by identical ISs in different sequence contexts led to the following conclusions: (i) transcription efficiency depends on the location of the IS in the defective interfering virus genome, (ii) downstream ISs have a negative effect on transcription levels from upstream ISs, and (iii) upstream ISs have little or no effect on downstream ISs. The observation that a downstream IS downregulates the amounts of sg RNA produced by an upstream IS explains why the smaller sg RNAs are, in general, produced in larger quantities than the larger sg RNAs. Our data are consistent with coronavirus transcription models in which ISs attenuate transcription. In these models, larger sg RNAs are synthesized in smaller amounts because they encounter more attenuating ISs during their synthesis.

Animals↗
PubMed1977

Wheat embryo ribonucleates. IX. Generation of N2-dimethylguanylate when bulk wheat embryo tRNA is used as substrate for wheat embryo S-adenosylmethionine-tRNA methyltransferases, in vitro.

Although the cellular ribonucleates in normally growing cells are virtually saturated with respect to their customary complement of methyl substituents, it has often been reported that 'marginal' levels of (homologous) methylation can be detected when ribonucleates and enzymes from the same source material are incubated, together with S-adenosylmethionine, in vitro. Experiments were designed to acquire new insights that might be useful for circumscribing the number of possible interpretations that could be advanced to account for the introduction of 'supernumerary' methyl groups during (homologous) methylation of wheat RNA by wheat enzymes, in vitro. For a large fraction of the supernumerary methyl groups that can be introduced into wheat RNA, in vitro, it was not possible to adduce convincing evidence in support of the view that any appreciable quantity of methyl groups is ever introduced at these same sites, in vivo. The possibility that these supernumerary methyl groups might have transient existence, in vivo, and the potential physiological significance of any such occurrence are dealt with as part of a more general discussion of the experimental findings.

Guanosine↗
PubMed1979

Cloning and characterization of ribosomal RNA genes from wheat and barley.

Wheat and barley DNA enriched for ribosomal RNA genes was isolated from actinomycin D-CsCl gradients and used to clone the ribosomal repeating units in the plasmid pAC184. All five chimeric plasmids isolated which contained wheat rDNA and eleven of the thirteen which had barley rDNA were stable and included full length ribosomal repeating units. Physical maps of all length variants cloned have been constructed using the restriction endonucleases Eco Rl, Bam Hl, Bgl II, Hind III and Sal I. Length variation in the repeat units was attributed to differences in the spacer regions. Comparison of Hae III and Hpa II digestion of cereal rDNAs and the cloned repeats suggests that most methylated cytosines in natural rDNA are in -CpG-. Incomplete methylation occurs at specific Bam Hl sites in barley DNA. Detectable quantities of ribosomal spacer sequences are not present at any genomic locations other than those of the ribosomal RNA gene repeats.

Base Sequence↗
PubMed1977

DNA-dependent RNA polymerase from T8 Guerin tumor.

DNA-dependent RNA polymerases from nuclei of T8 Guerin tumor were studied. Two enzymes were purified several hundred times by the use of ammonium sulfate precipitation, DEAE-cellulose and phosphocellulose chromatography. One of them belongs to A(I) RNA polymerases and the second to B(II) as was established from their metal and ionic strength requirements. activity in the presence of native and denatured DNA and the resistance to a-amanitin inhibition. The quantity of class A enzyme was increased compared to B, a fact observed with most neoplastic tissues so far studied. This increase of the polymerase responsible for ribosomal RNA synthesis could probably be related to malignant transformation in animals.

Adenocarcinoma↗
PubMed2005

LacI-mediated sequence-specific affinity purification of plasmid DNA for therapeutic applications.

Affinity purification of plasmid DNA is an attractive option for the biomanufacture of therapeutic plasmids, which are strictly controlled for levels of host protein, DNA, RNA, and endotoxin. Plasmid vectors are considered to be a safer alternative than viruses for gene therapy, but milligram quantities of DNA are required per dose. Previous affinity approaches have involved triplex DNA formation and a sequence-specific zinc finger protein. We present a more generically applicable protein-based approach, which exploits the lac operator, present in a wide diversity of plasmids, as a target sequence. We used a GFP/His-tagged LacI protein, which is precomplexed with the plasmid, and the resulting complex was immobilized on a solid support (TALON resin). Ensuing elution gives plasmid DNA, in good yield (>80% based on recovered starting material, 35-50% overall process), free from detectable RNA and protein and with minimal genomic DNA contamination. Such an affinity-based process should enhance plasmid purity and ultimately, after appropriate development, may simplify the biomanufacturing process of therapeutic plasmids.

Bacterial Proteins↗
PubMed1983

Cytological distribution of chorionic gonadotropin subunit and placental lactogen messenger RNA in neoplasms derived from human placenta.

Normal trophoblast of the human placenta elaborates at least two major protein hormones, chorionic gonadotropin (hCG), and placental lactogen (hPL). There are several gestational trophoblastic diseases of the placenta called hydatidiform mole, invasive mole, and choriocarcinoma. Molar and choriocarcinoma tissues characteristically synthesize large amounts of hCG and small quantities of hPL. To examine the role of trophoblast differentiation in the expression of the hCG and hPL genes, we studied the cytological distribution of their messenger RNA (mRNA) in tissue sections of human hydatidiform mole and choriocarcinoma by in situ hybridization. Histologically, these tissues are in different stages of cellular differentiation. In normal placenta, hCG alpha and -beta mRNA can be localized to some cytotrophoblasts and primarily to the syncytium, whereas hPL mRNA appears only in the syncytial layer. In hydatidiform mole, which still retains placental villous morphology, the hPL gene and hCG alpha and -beta genes are expressed but are poorly localized because of the admixture of cyto- and syncytiotrophoblasts. By contrast, choriocarcinoma, which is devoid of placental villous pattern but in which the cyto- and syncytiotrophoblast-like components are distinguishable, expresses hCG alpha and -beta in the syncytial-like areas but little, if any, hPL. These results suggest that a certain level of trophoblast differentiation, such as villous formation, is associated with hPL expression, while the hCG alpha gene and the hCG beta gene can be expressed in more disorganized tissues that contain cytotrophoblastic elements.

Cell Differentiation↗
PubMed1983

Large-scale isolation of plasmid DNA and purification of lambda phage DNA using hydroxylapatite chromatography.

A rapid and relatively simple procedure for purifying large quantities of plasmid DNA is described. Plasmid thus purified contains no detectable chromosomal DNA and little RNA or protein. The procedure combines alkaline denaturation and hydroxylapatite chromatography and utilizes an improved method of separating DNA from RNA. It was observed that the phosphate concentrations at which previously bound DNA as well as RNA elute from hydroxylapatite changed markedly as a function of urea concentration. In the presence of urea concentrations higher than 4 M, the ranges of phosphate concentration over which DNA and RNA elute show no overlap. This permits efficient washing of hydroxylapatite-bound DNA under conditions which should remove all bound RNA. lambda Phage DNA is also easily eluted from hydroxylapatite under the conditions used.

Adsorption↗
PubMed2004

Synchronous sperm retrieval and sperm washing in an intracytoplasmic sperm injection cycle in an azoospermic man who was positive for human immunodeficiency virus.

OBJECTIVE: To present the first reported case of synchronous sperm retrieval followed by sperm washing before an intracytoplasmic sperm injection (ICSI) cycle in an HIV-positive azoospermic man. DESIGN: Case report. SETTING: Assisted reproduction center. PATIENT(S): A 40-year-old HIV-positive man with obstructive azoospermia due to vasal aplasia. INTERVENTION(S): Synchronous sperm retrieval, sperm washing, nucleic acid-based sequence amplification testing, and intracytoplasmic sperm injection. MAIN OUTCOME MEASURE(S): Successful sperm retrieval sufficient for sperm washing and fertilization. RESULT(S): Sufficient quantity of spermatozoa for washing was obtained at epididymal aspiration. After the wash, HIV ribonucleic acid (RNA) was undetectable with nucleic acid-based sequence amplification testing, enabling injection of oocytes collected after routine gonadotropin superovulation. Of seven oocytes collected from the 39-year-old woman partner, six were injected and five fertilized (83%). Three embryos were transferred on day 2. The pregnancy test was negative on this occasion. CONCLUSION(S): This case demonstrates that sperm washing can be applied in cases of sperm retrieval where sperm volume and density is low, allowing the treatment of azoospermic HIV-positive men.

Adult↗
PubMed1995

Complement regulation in the rat glomerulus: Crry and CD59 regulate complement in glomerular mesangial and endothelial cells.

The complement regulators, decay accelerating factor, membrane cofactor protein, and CD59 are present in human glomeruli. Crry is the rodent analogue to the former two proteins. In this study, we examined complement regulation in cultured rat glomerular endothelial cells (GEnC) and mesangial cells (MES). Immunoprecipitation of 125I-labeled membrane proteins and Western blotting studies were performed with anti-Crry and anti-CD59. In both GEnC and MES, Crry was present as 53, 65, and 78 kD proteins. The 20 kD CD59 was apparent in GEnC. CD59 was also present in MES, but in relatively smaller quantities. By Northern analyses, 1.8 kb CD59 mRNA was present in GEnC as well as in RNA from isolated rat glomeruli. mRNA for Crry was present in both GEnC and MES as 2.2 kb species. The functional significance of these proteins was evaluated next. Anti-Thy 1.1 IgG was used to activate the complement classical pathway in MES. To inhibit the function of the complement regulators, anti-CD59 and/or anti-Crry F(ab')2 antibodies were added with anti-Thy 1.1. Inhibition of Crry function led to enhanced cytotoxicity, while there was no effect when CD59 function was inhibited. The complement alternative pathway was studied by adding complement in Mg-EGTA buffer. Inhibition of Crry led to productive alternative pathway activation, which was accentuated by anti-CD59 when Crry was incompletely inhibited. Alternative pathway regulation was also evaluated in GEnC. Inhibition of CD59 function alone had no effect in GEnC, while inhibition of Crry led to significant cytotoxicity from alternative pathway activation. Under conditions in which Crry was inactive, inhibition of CD59 further enhanced cytotoxicity. Therefore, Crry is present in both GEnC and MES and restricts the complement alternative pathway in both cell types. Crry also regulates the classical pathway in MES. CD59 is present and functionally active in GEnC, while it appears to have a minor role in MES.

Animals↗
PubMed1973

Studies of nuclei separated by zonal centrifugation from liver of rats treated with thioacetamide.

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.

Acetamides↗
PubMed1976

Isolation of single stranded DNA related to the transcriptional activity of animal cells.

Single stranded DNA (s.s.DNA) comprising 1-2% of the total nuclear DNA was isolated by an improved method of hydroxyapatite chromatography from native nuclear DNA3 of embryonic chick cells, labeled for several cell generations with 3H-thymidine. Small quantities of 3H-DNA were annealed with a large excess of unlabeled DNA or polysomal RNA from chick embryos. Hybridization kinetics (monitored by the use of SI nuclease digestion, hydroxyapatite chromatography and thermalfusion), indicated that s.s.DNA belongs to the non repetitious fraction of the cell genome. One third represents DNA sequences engaged in the transcription of messenger RNA's.

Animals↗
PubMed1976

Regulatory mechanism of simian virus 40 gene expression in permissive and in nonpermissive cells.

Primary or continuous lines of mouse cells (3T3) are nonpermissive for simian virus 40 (SV40). Abortively infected cells synthesize tumor antigen (T antigen but not viral DNA and virus capsid protein (V antigen). V antigen, however, was obtained when SV40 DNA was injected into 3T3 cells. This late gene expression also appears to be correlated with the quantity of injected DNA molecules per 3T3 cell. T antigen formation can be detected after microinjection of only 1 to 2 DNA molecules, but the intensity of intranuclear T antigen fluorescence is significantly brighter with injection of higher concentrations of viral DNA. In permissive cells (TC7), early and late SV40 gene expression is directly related to the number of injected molecules. Microinjection of 1DNA molecule induced T and V antigen formation with the same efficiency as microinjection of 2,000 to 4,000 molecules. The question of weather late SV40 gene expression is directly related to the quantity of an early virus-specific product was approached by microinjection of early SV40 complementary RNA together with small amounts of viral DNA. V antigen was obtained in a high proportion of recipient 3T3 cells at conditions where microinjection of viral DNA alone induced T but not V antigen synthesis.

Animals↗
PubMed1978

Meiotic maturation of mouse oocytes in vitro: protein synthesis in nucleate and anucleate oocyte fragments.

Nucleate and anucleate fragments of mouse oocytes have been isolated following treatment of fully grown oocytes with cytochalasin B. The nucleate oocyte fragments resume meiosis in vitro, progressing from dictyate of the first meiotic prophase to metaphase II ('meiotic maturation'), and exhibit all of the changes in protein synthesis normally associated with meiotic maturation of mouse oocytes. The anucleate oocyte fragments also undergo certain of the changes in protein synthesis associated with meiotic maturation, despite the absence of nuclear progression. These results suggest that the acquisition of meiotic competence (i.e. the ability to undergo meiotic maturation) during growth of the mammalian oocyte is due to changes in the quality, rather than the quantity, of cytoplasm and that the reprogramming of protein synthesis during meiotic maturation is directed by RNA templates already present in the cytoplasm. The behaviour of anucleate oocyte fragments is discussed in terms of the proposed role for nucleoplasm in the initiation of changes in protein synthesis during meiotic maturation of mouse oocytes.

Animals↗
PubMed1983

Time course of biochemical and histological alterations following a single feeding of carbon tetrachloride to mice.

Carbon tetrachloride (0.02, 0.5 or 1.5 ml/kg) was given through a stomach tube to female C57BL/6J mice. Liver weights, the quantities of nine liver (protein, free glucose, glycogen, triglyceride, phospholipid, free and esterified cholesterol, DNA and RNA) and three blood components (glucose and free and esterified cholesterol), and SGOT activities were measured at 15, 24, 39, 48, 63, 87, 159 or 327 hr after carbon tetrachloride feeding. Livers were examined histologically. Chemical components of the liver and blood changed generally in a dose-related manner, but more slowly in the larger-dose groups. The biochemical alterations were reflected well in the histological findings. Degenerative and regenerative findings occurred later in the larger-dose animals. Histological changes appeared first in the midzones in the 0.5 and 1.5 ml/kg groups.

Animals↗