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PubMed1999

Use of bovine viral diarrhoea virus as an internal control for amplification of hepatitis C virus.

BACKGROUND AND OBJECTIVES: Screening for hepatitis C virus (HCV) by polymerase chain reaction (PCR) will be mandatory for screening blood and plasma donors in Europe and elsewhere. This study describes an internally controlled, highly sensitive PCR method designed for screening blood donations in pools. MATERIAL AND METHODS: RNA extracted from bovine viral diarrhoea virus (BVDV) was used as an internal control to monitor the efficiency of extraction, reverse transcription and amplification steps in HCV PCR. RESULTS: Sensitivity of PCR for single molecules of HCV in the presence of 33 genome equivalents of BVDV RNA was achieved by reducing the efficiency of BVDV amplification. BVDV could be recovered at high efficiency from large volume pools (2-5 ml) by ultracentrifugation and by the NucliSens extraction method. CONCLUSION: Detection of BVDV validates the extraction, reverse transcription and amplification methods used for HCV detection in plasma pools and provides valuable quality assurance for negative results.

Animals↗
PubMed1969

Responses to immunization in the thymus of the adult mouse.

The rate of synthesis of RNA in the thymus glands of adult mice increased after immunization with sheep red blood cells (SRBC). The specific activity of some fractions of RNA, separated first by density gradient centrifugation and then by polyacrylamide gel electrophoresis, was 16-fold higher on day 3 after immunization than control mice not injected. RNA synthesis in the thymus was inhibited by rabbit anti-mouse thymus serum, injected along with antigen. A material was found in RNA extracts from the thymus glands of mice immunized with SRBC which converted a small proportion of either spleen cells or peritoneal cells from nonimmunized mice to form sheep cell hemolysins. Neither extracts from the glands of nonimmunized mice nor the livers of immunized mice were active. Extracts from the thymus glands of mice immunized with rabbit red blood cells (RRBC) were inactive and activity was destroyed by ribonudease. The residual antigen content was not determined. Biologically active extracts from the thymus had a different electrophoretic mobility from active extracts from the spleen.

Animals↗
PubMed1986

Expression of the major heat shock protein (hsp 70) family during early mouse embryo development.

Stress or heat shock proteins (hsp) are synthesized by most cells in response to adverse environmental conditions. In mammalian cells, the major proteins synthesized in response to stress have relative molecular weights (Mr) in the range of 68 to 74 kilodaltons (kD) and are encoded by a small multi-gene family collectively referred to as hsp 70 genes. In unfertilized mouse eggs, no members of the hsp 70 family appear to be synthesized under normal or stressful (heat shock) conditions. At the two-cell stage, two proteins with Mr = 74 kD and Mr = 70 kD are expressed as a consequence of developmentally activated transcription of these hsp 70 genes. No stress-induced synthesis in response to heat shock is observed at this stage. At the eight-cell stage, constitutive synthesis of the 70-kD protein continues, but, as in the two-cell embryo, no heat shock induced synthesis of a novel heat shock protein is observed. By the blastocyst stage, however, an inducible protein with Mr = 68 kD is synthesized in response to heat shock in addition to constitutive synthesis of the 70-kD protein. The constitutively synthesized cognate proteins are coded for by a set of mRNAs about 2,000 nucleotides in length. The induced hsp 68 proteins are coded for by mRNAs of larger size (about 2,600 nucleotides). Only the smaller mRNA class is detectable on Northern blots of RNA extracted from control or heat shock cleavage stage embryos and control blastocysts. As predicted from protein synthetic studies, both classes are resolved in RNA preparations derived from heat-shocked blastocysts.

Animals↗
PubMed1980

Survival, DNA synthesis and ribosomal RNA transcription in monkey kidney cells treated by formaldehyde.

Separate cultures of CV-1 cells were exposed for 15 min to 1-16 mM formaldehyde (FA) at various time intervals before labeling with [3H]uridine. The labeled RNA extracted from whole cells was analyzed by polyacrylamide gel electrophoresis. Results indicated that FA produced transcription-terminating lesions in DNA which depressed RNA synthesis, gave rise to shortened RNA chains and modified the expression of transcription-linked genes. These lesions were efficiently repaired since a recovery of RNA transcription, with disappearance of the initially observed alterations, rapidly occurred during post-treatment incubation of cells. Cycloheximide (5 micrograms/ml) strongly inhibited this recovery, whereas fluorodeoxyuridine (10(-5) M) was without effect. The rate of semi-conservative DNA synthesis, measured autoradiographically by thymidine incorporation, as well as the number of cells performing DNA replication, fell to zero after 15-min exposure to FA concentrations greater than 2 mM. Both parameters recovered subnormal levels during a 24-h incubation after treatment with FA concentration up to 8 mM, in agreement with the high survival observed. Unscheduled DNA synthesis was not detectable during the restoration of DNA and RNA synthesis.

Animals↗
PubMed1987

Detection of flavivirus RNA in infected cells using photobiotin-labelled hybridization probes.

Ten plasmids containing viral cDNA inserts of portions of the dengue virus type 2 (DEN-2) or Kunjin virus (KUN) genomes were biotinylated using photobiotin acetate and used as probes for the detection of flavivirus RNA in infected Vero cells. The viral cDNA inserts ranged in length from 0.19 to 2.7 kilobase pairs, and represented segments of the flavivirus genome coding for structural and nonstructural proteins. In spot hybridization assays (hybridization at 60 degrees C) with RNA extracted from cells infected with one of fourteen different flaviviruses or Semliki Forest virus, all DEN-2 and KUN probes hybridized specifically with RNA from cells infected with DEN-2 or KUN, respectively. At the reduced stringency of lower temperatures, specific hybridization to homologous viral RNA was still a feature of the probes, and only limited cross-hybridization to the RNA of some other flavivirus species was detected.

Animals↗
PubMed1997

16S mitochondrial ribosomal RNA degradation is associated with apoptosis.

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.

Animals↗
PubMed1990

A new recombinant DNA strategy for the molecular cloning of rare membrane proteins.

We have constructed a cDNA library in the plasmid expression vector pUEX enriched in sequences encoding membrane proteins. The procedure involved positive selection of sequences common to two different rat tissues (thus excluding tissue-specific mRNA) followed by positive selection between this material and RNA extracted from membrane bound polysomes (thus excluding cytoplasmic proteins). The resultant library prepared from rat kidney cDNA hybridized with rat liver poly(A)+ RNA, contained 30,000 clones and was shown to be enriched in cDNAs encoding membrane proteins. Seventeen clones selected because they encode large fusion proteins were shown to be single copy in the library, and not present in nucleotide data banks. Thus the strategy is particularly suitable for cloning low abundance cDNAs encoding membrane proteins.

Animals↗
PubMed1968

RNA synthesis in Chinese hamster cells. I. Differential synthetic rate for ribosomal RNA in early and late interphase.

The incorporation of methionine-methyl-(14)C into 18S ribosomal RNA of cultured Chinese hamster ovary cells in early and late interphase has been determined by zone-sedimentation analysis of phenol-extracted RNA preparations. Synchronized cell cultures were prepared for these studies by thymidine treatment and by mechanical selection of mitotic cells. The specific activity of 18S RNA labeled in late interphase was found to be 1.1-1.2 times that of 18S RNA labeled in early interphase. Upon correction for increase in RNA mass, the rate of methylation of 18S RNA in late interphase is about 1.9 times that in early interphase.

Animals↗
PubMed1982

In Vitro Synthesis and Processing of Wheat alpha-Amylase : TRANSLATION OF GIBBERELLIC ACID-INDUCED WHEAT ALEURONE LAYER RNA BY WHEAT GERM AND XENOPUS LAEVIS OOCYTE SYSTEMS.

Wheat (Triticum aestivum) RNA was used to program synthesis of the alpha-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as alpha-amylase by immunoprecipitation with rabbit anti-alpha-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic alpha-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of alpha-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of alpha-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the alpha-amylase protein to a form which co-migrated with authentic alpha-amylase purified from malted wheat and with the protein synthesized in oocytes.

Journal Article↗
PubMed1984

Translation of messenger RNA from rat epididymis and identification of poly(A)RNA coding for acidic epididymal glycoprotein.

Different proteins are secreted by the various regions of rat epididymis. We have examined the messenger RNA dependence of this varied gene expression by cell-free translation of poly(A)RNA extracts from initial segment, caput plus corpus, and cauda. Labeled translation products were analyzed by polyacrylamide gel electrophoresis under denaturing conditions. Poly(A)RNA from initial segment coded for several unique bands of labeled protein, including a 23,000 MW protein that may correspond to an initial segment protein reported previously to be regulated by testicular fluid factors. Messenger RNA encoding 20,000 MW protein believed to be alpha-lactalbumin, was most abundant in caput but also present in initial segment. Acidic epididymal glycoprotein (AEG) was identified previously by immunoperoxidase staining in epithelial cells of caput, corpus, and cauda. AEG was not readily identified on electrophoresis of total translated proteins, but when concentrated by immunoprecipitation with purified AEG antibody prior to electrophoresis, AEG appeared in both caput plus corpus, and in cauda poly(A)RNA translations.

Animals↗
PubMed1970

Foot-and-mouth disease virus-induced alterations of baby hamster kidney cell macromolecular biosynthesis: inhibition of ribonucleic acid methylation and stimulation of ribonucleic acid synthesis.

The kinetics of ribonucleic acid (RNA) and protein synthesis and RNA methylation were examined after foot-and-mouth disease virus (FMDV) infection of baby hamster kidney cells. The synthesis of RNA extracted from the whole cells was stimulated two- to threefold above the control level of synthesis. This increased rate was attributed to viral RNA synthesis. The inhibition of host RNA methylation was concomitant with but more pronounced than protein synthesis inhibition. The methylation of transfer RNA was initially inhibited by virus infection, but rose to within 70 to 80% of the control level just prior to the production of maximal amounts of virus-specific RNA polymerase. Cycloheximide studies showed that rapid cessation of protein synthesis did not result in the immediate cessation of RNA methylation. A comparison between the kinetics of inhibition of these processes by cycloheximide and FMDV infection suggests that FMDV selectively inhibits RNA methylation.

Animals↗
PubMed1994

Expression of a rabbit renal ascorbic acid transporter in Xenopus laevis oocytes.

We examined the expression of renal ascorbic acid transporter(s) in Xenopus laevis oocytes after microinjection of cells with poly(A)+ RNA extracted from rabbit kidney cortex. Concomitant expression of the Na+-glucose cotransporter served as a control in these studies. Injection of poly(A)+ RNA into oocytes produced over a fivefold increase in the uptake of [14C]ascorbic acid (570 microM) compared with water-injected cells. Size fractionation of the kidney cortex mRNA by sucrose gradient revealed that the mRNA species that induced ascorbic acid transporter expression in oocytes was present in a fraction centered around 2.0 kilobases (kb) and had a size range of 1.8-3.1 kb. Injection of the active fraction into oocytes produced a > 40-fold increase in ascorbic acid uptake compared with water-injected controls. Expression of ascorbic acid transporter(s) was noticeable as early as 2 days after injection and was maximal after 7 days; it was also dependent on the amount of mRNA injected into oocytes. The induced uptake of [14C]ascorbic acid after injection of mRNA into oocytes was 1) Na+ dependent, as indicated by the almost complete lack of transport on removal of Na+ from the incubation medium; 2) significantly inhibited by unlabeled ascorbic acid and its structural analogue isoascorbic acid but not by D-glucose; and 3) saturable as a function of increasing the substrate concentration in the incubation medium (100-1,000 microM), with an apparent Km of 258 +/- 72.5 microM and a maximum velocity of 29.6 +/- 2.8 pmol.oocyte-1.2 h-1. These data demonstrate that X. laevis oocytes are a suitable system to functionally express the mammalian renal ascorbic acid transporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

Epidermal growth factor, epidermal growth factor receptor, and transforming growth factor-alpha in human hyperplastic prostate tissue: expression and cellular localization.

It is widely accepted that polypeptide growth factors are involved in the growth and development of normal and neoplastic human prostate. It has been previously reported that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) receptors are present in the human hyperplastic prostate tissue (BPH). To add information on the mechanism of action of EGF and transforming growth factor-alpha (TGF alpha), a peptide correlated to EGF, and the EGF receptor (EGF-R) in the human prostate, we studied the expression and cellular localization of messenger ribonucleic acid (RNA) encoding EGF, EGF-R, and TGF alpha in BPH tissue. Reverse transcriptase-PCR of total RNA extracted from BPH tissues documented the presence of specific transcripts for EGF, EGF-R, and TGF alpha. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that EGF, EGF-R, and TGF alpha messenger RNAs were mainly localized in the epithelial cells. Immunprecipitation and Western blot analysis showed that BPH tissue contained the corresponding proteins, EGF and TGF alpha. Our findings provide additional support for the idea that EGF and TGF alpha may be considered specialized symbols in the language of cell-cell interactions and for the hypothesis that in the human prostate they seem to act in an autocrine fashion.

Epidermal Growth Factor↗
PubMed2006

Community dynamics of methanotrophic bacteria during composting of organic matter.

In this study, we describe the effects of composting on the diversity, abundance and activity of the methanotrophic community present in the compost. Composting was allowed to proceed for 10 weeks in an in-vessel reactor. Self-heating capacity (Rottegrad) indicated that compost maturity was reached after 4 weeks. After 6 weeks, a second thermophilic phase was induced by manually increasing temperature to investigate whether or not the methanotrophs shifted back to the thermophilic population. Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) with primers specific for type I methanotrophs of 16S rDNA and 16S rRNA were used to characterize the composition of the microbial community. Cluster and diversity analyses of RNA DGGE patterns were more sensitive than those of DNA DGGE patterns, and revealed that mesophilic and thermophilic methanotrophic communities could be differentiated. Moreover, it was seen that the diversity of the community was low during the thermophilic phase and increased during the final maturation phase. Real-time PCR analysis was also performed on the DNA and RNA extracts and showed no changes in the abundance of type I methanotrophs during the composting process (10(9) DNA copies/g compost). However, RNA-related activity did change, with the lowest activity (10(7) cDNA copies/g compost) observed during the thermophilic phase, subsequently increasing to its maximum value (10(9) cDNA copies/g compost), and finally decreasing during the maturation phase. This study confirmed the population dynamics, as seen for general groups such as bacteria and fungi during composting, for a very specific and sensitive group of bacteria, it is the type I methanotrophs.

Bacteria↗
PubMed2006

Comparison of in vitro and in vivo reference genes for internal standardization of real-time PCR data.

Real-time PCR is a powerful technique for gene expression studies, which have become increasingly important in a large number of clinical and scientific fields. The significance of the obtained results strongly depends on the normalization of the data to compensate for differences between the samples. The most widely used approach is to use endogenous reference genes (housekeeping genes) as internal standards. This approach is controversially discussed in the literature because none of the reference genes is stably expressed throughout all biological samples. Therefore, candidate reference genes have to be validated for each experimental condition. In our studies, we introduced and evaluated an in vitro synthesized reference cRNA for internal standardization of relative messenger RNA (mRNA) expression patterns. This reference, consisting of the in vitro transcribed coding sequence of aequorin, a jellyfish protein, was incorporated in the extracted RNA. The experimental significance of this approach was representatively tested for the expression of the neurotrophin-3 mRNA in distinct regions of mouse brains. A comparison to three stably expressed reference genes [beta-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and hypoxanthine phosphoribosyl-transferase 1 (HPRT1)] gave evidence that the spiking of template RNA with in vitro transcribed cRNA is a valuable tool for internal standardization of real-time PCR experiments.

Actins↗
PubMed2000

Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation.

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aalpha-chain gene, only 2 missense mutations in the C-terminal domain of the Bbeta-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G-->A transition at position +5 of intron 1 of the gamma-chain gene. The predicted mutant fibrinogen gamma-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal role of the identified mutation, we prepared expression vectors containing a region of the fibrinogen gamma-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1. Transient transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen gamma-chain gene causing afibrinogenemia and indicates that, in addition to the Aalpha and Bbeta-chain genes, the gamma-chain gene must also be considered in mutation screening for afibrinogenemia.

Afibrinogenemia↗
PubMed1977

Molecular changes in lymph node tissue after skin heterografts.

Changes in the rat lymph node RNA base composition and in ribosomal aggregation induced by heterologous skin graft have been characterized by the base composition analysis of the phenol extractable RNA, by the density gradient centrifugation of the ribosomal particles and by amino acid incorporation in the newly synthetized protein. Following skin grafts, the changes in base composition of total RNA in lymph . nodes were mainly due to the synthesis of ribosomal type RNA. Ribosomes were assembled as single units or as polyribosomes. The present findings also show an increase in amino acid incorporation by polyribosomes after the first graft reaction.

Amino Acids↗