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At least 955 records · Page 53Linked to original sources
PubMed1988

Analysis of von Willebrand factor mRNA from the lung of pigs with severe von Willebrand disease by using a human cDNA probe.

To examine the control of porcine von Willebrand factor (vWF) biosynthesis we cloned human vWF complementary DNA (cDNA) and investigated the expression of the vWF gene in lungs from normal pigs and pigs with severe von Willebrand's disease (vWD). Recombinant clones spanning approximately 90% of human vWF cDNA were identified in a lambda gt10 human lung cDNA library by screening with oligonucleotides. One clone spanning nucleotides 960 to 3,240 of human vWF cDNA was used to investigate the steady-state levels of vWF mRNA in lungs from normal pigs and from pigs phenotypically determined to be homozygous for vWD. This clone hybridized with genomic DNA from pig leukocytes when Southern blots were processed under very stringent conditions; therefore, human cDNA clones were considered valid probes to detect porcine mRNA. Northern blot analysis of total RNA from normal pig lung and human umbilical vein endothelial cells identified the vWF mRNA as a molecular species of approximately 9.0 kilobases (kb). A very faint to undetectable band at 9.0 kb in total RNA from lungs of vWD pigs suggested a decreased rate of transcription of the vWF gene. Sucrose density gradient centrifugation of RNA from the vWD pigs confirmed by Northern analysis that the high-molecular weight fractions contained vWF mRNA and at the same size as normal pig mRNA. Dot blot hybridization analysis of vWF and actin mRNA processed under stringent conditions demonstrated that the relative ratio of vWF mRNA to actin mRNA in the vWD pigs varied from 21% to 41% of the ratio observed in normal pigs. Because the amount of vWF mRNA is not correlated to the amount of vWF activity or antigen in plasma of vWD pigs we conclude that posttranscriptional events are also probably involved in abnormal expression of vWF in these animals.

Animals↗
PubMed1998

Identification of sex hormone receptors in human and rabbit ligaments of the knee by reverse transcription-polymerase chain reaction: evidence that receptors are present in tissue from both male and female subjects.

Gender-related factors have been attributed to observed differences in the rate of injury to ligaments (e.g., anterior cruciate ligament) between male and female subjects. These differences may be a result of unique regulatory mechanisms within the tissue in response to the sex hormones estrogen and progesterone. These hormones, when bound to specific intracellular receptors (estrogen receptor and progesterone receptor, respectively), modulate gene expression within hormone-responsive tissue. The purpose of this study was to evaluate the expression of the estrogen and progesterone receptors in ligament tissue from male and female rabbits and humans by the sensitive molecular technique of reverse transcription-polymerase chain reaction. Total RNA was extracted from human anterior cruciate ligament tissue and from medial cruciate ligament, anterior cruciate ligament, patellar tendon, and synovium tissue of the New Zealand White rabbit by the newly developed TRIspin method. The total RNA was reverse transcribed and analyzed by polymerase chain reaction to assess the expression of estrogen and progesterone receptors. Our results demonstrate that estrogen and progesterone receptor transcripts are expressed in ligament tissue of male and female rabbits and humans and that alterations in receptor expression occur in ligaments during pregnancy. In the human samples, only a small percentage of the estrogen receptor appeared to be a nonfunctional mRNA splice variant, and the predominant form contained the estrogen-binding domain.

Alternative Splicing↗
PubMed1996

Comparison of four methods for isolating large mRNA: apolipoprotein B mRNA in bovine and rat livers.

The effectiveness of four methods for isolating large-size mRNA (14 kb) encoding for hepatic bovine apoprotein B (apo B) was compared. Total RNA of liver (positive controls) and lung (negative controls) samples taken in bovine and rat was extracted using the methods of Chirgwin et al., Chomczynski and Sacchi, Cathala et al., and RNAzol. The integrity of total RNA extracted by the four methods was demonstrated by electrophoresis on 1% agarose gel and staining with ethidium bromide. Yield of extraction was two- to four-fold higher for all samples with the methods of Chirgwin et al. and RNAzol than that with the other methods. By dot blot analysis, apo B mRNA in bovine and rat livers was revealed preferentially with Chirgwin et al. and RNAzol methods. By Northern blot analysis, a single band corresponding to apo B100 mRNA was shown in bovine and rat livers only with the method of Chirgwin et al., whereas a band corresponding to a medium-size mRNA (2.6 kb) encoding for rat phosphoenolpyruvate carboxykinase was revealed in rat liver samples with the four methods. These results showed the favorable role of guanidium isothiocyanate and guanidium hydrochloride used in the extraction and the purification of RNA, respectively, especially in the case of high-molecular-weight and/or low-represented RNA such as apo B mRNA in bovine liver.

Animals↗
PubMed1995

Induction of inducible nitric oxide synthase expression by neopterin in vascular smooth muscle cells.

The pteridine compounds neopterin and 7,8-dihydroneopterin serve as valuable indicators for the stimulation of the cellular immune system. Whether they exhibit distinct biochemical functions in the immunological process is at present under discussion. We show that neopterin, but not 7,8-dihydroneopterin, is a stimulus for iNOS gene expression in rat vascular smooth muscle cells in vitro. At a concentration of 20 microM, neopterin leads to an iNOS mRNA expression of 2.5 amol iNOS cDNA/micrograms total RNA. When cells were coincubated with 20 microM neopterin and 5 micrograms/ml lipopolysaccharide derived from Escherichia coli, at least an additive effect on iNOS mRNA expression could be detected (iNOS cDNA concentration was 5.0 amol/micrograms total RNA). We speculate that neopterin enhances the macrophage-induced extracellular toxicity. This might be of relevance in situations associated with excessive release of cytokines, neopterin, and nitric oxide, as observed in septic shock.

Animals↗
PubMed2001

Region-specific transcriptional activity in the genome of Streptomyces coelicolor A3(2).

Transcriptional analysis of microbial genomes is an important component of functional genomics. Strategies such as hybridization of labeled total RNA against ordered clone libraries or differential-display approaches have already been carried out to identify expressed genes. We describe here an additional method which applies subtractive hybridization between genome-specific DNA and total RNA followed by a PCR approach to identify expressed microbial genes. With the new strategy, the expression of genes in the terminal regions of the linear Streptomyces coelicolor A3(2) chromosome and the accessory linear plasmid SCP1 was analyzed. The results indicate that the method is useful for the identification of expressed genes in actinomycetes and other microbial systems. We demonstrate for the first time that at least 24 genes in the chromosome end regions (silent regions) of S. coelicolor are actively expressed. In addition, several expressed SCP1 genes were identified, including a gene which shows high similarity to microbial dnaN genes and which seems to play a role in SCP1 maintenance.

Bacterial Proteins↗
PubMed1975

[Kinetics of the synthesis of nucleic acids in haploid and diploid loach embryos].

Al kinetic analysis of incorporation of the mixture of 3H-nucleosides in the nucleic acid fractions was carried out to examine the mechanisms of compensation of the genetic material deficiency. Both the haploid and diploid embryos of the loach (Misgurnus fossilis L.) were analyzed. When comparing the DNA and RNA syntheses, the level of phosphorylation (nucleotide pool) in the both genetic variants was under control. The rate of incorporation of the labelled nucleosides in DNA was shown to be higher in haploids at the early developmental stages than in diploids but later it became the same. The increased level of DNA replication in the early haploid embryos was due to the compensatory increase of the cell number in them as compared with the diploid ones. The rate of total RNA synthesis corrected by the differences in the rate of nucleoside phosphorylation varied directly with the degree of ploidy at the blastula stage; at the gastrula stage the value of RNA synthesis per haploid genome was compensated, and at the stage of organogenesis the production of total RNA, as calculated per cell, became in haploids even higher than in diploids. The data obtained suggest the essential changes in the patterns of RNA synthesis control during development.

Age Factors↗
PubMed1993

Non-radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles.

A method was established for measuring molecule numbers of three different myosin heavy chain (MHC) mRNA isoforms in total RNA preparations. The quantification was based on a combination of primer-directed reverse transcriptase and polymerase chain reactions with 5'-digoxigenin-labeled oligonucleotides, using external standards. The sensitivity of the method allowed the quantitation of mRNA amounts down to the range of 1,000 molecules (detection limit 50 molecules). The numbers determined for eight different rabbit muscles are in the range of 10(3)-10(9)/micrograms total RNA. In soleus muscle, the value of 1.11 x 10(9) MHCI mRNA molecules corresponds to approximately 8% of the total mRNA. With reference to myonuclei, this amount corresponds to 1-2 x 10(4) molecules/nucleus. A quantitative comparison of the two fast MHC mRNA isoforms with the distribution of different MHC isoforms at the protein level indicates that one of these two fast sequences is specific to MHCIIb and the other to MHCIId. However, our data point to the existence of additional MHCIId mRNA subtypes.

Animals↗
PubMed1991

Primary demyelination induced by exposure to tellurium alters mRNA levels for nerve growth factor receptor, SCIP, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and myelin proteolipid protein in rat sciatic nerve.

Weanling rats fed a diet containing tellurium develop a peripheral neuropathy characterized by a highly synchronous primary demyelination; this demyelination is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis. Expression of mRNA for the major structural proteins of PNS myelin, myelin basic protein and P0, is coordinately down-regulated during the demyelinating phase and then up-regulated during the remyelinating phase (Toews et al., J. Neurosci. Res., 26 (1990) 501-507). We now report tellurium-induced alterations in gene expression for several proteins which are not major structural components of myelin in the peripheral nervous system. Expression of mRNA for nerve growth factor receptor in sciatic nerve was very low in control animals, but was markedly up-regulated after 3-5 days of exposure to tellurium, a time corresponding to the beginning of demyelination. Levels remained elevated during the subsequent period of remyelination. Expression of mRNA for SCIP (a presumptive transcription factor) was also up-regulated in sciatic nerve following tellurium exposure, with a time course similar to that for nerve growth factor receptor. When examined as a fraction of total RNA, steady-state mRNA levels for 2',3'-cyclic nucleotide 3'-phosphodiesterase and the myelin proteolipid protein were decreased during the demyelinating phase; however, this decrease could be largely accounted for by increased levels of total RNA. When analyzed on a 'per nerve' basis, steady-state mRNA levels for these two proteins were actually increased about 2-fold by 9 days after beginning tellurium exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed2000

[hBD-2 gene expression in nasal mucosa].

BACKGROUND: Chronic sinusitis is one of the frequent inflammatory diseases and has a complex pathogenesis. A substantial factor seems to be recurrent bacterial infections. Pseudomonas aeruginosa (PA) frequently can be found in nasal smears of patients with persistent sinus symptoms after sinus surgery. Lately a new antimicrobial peptide of epithelial origin, human Beta-Defensin-2 (hBD-2), with a strong antibacterial effect against PA could be identified within lesional skin scales of patients suffering psoriasis. Aim of this study was to investigate hBD-2-mRNA expression in nasal cells and tissue. METHODS: Total RNA was extracted from nasal polyps and turbinates following TRIzol protocol. Epithelial cells and fibroblasts of nasal human tissue were isolated and cultivated. The cells were stimulated with PA using different time points and different concentrations. Total RNA was isolated as mentioned above, reverse transcribed and amplified in a Semi-quantitative Reverse Transcriptase PCR (SQRT-PCR) with genespecific hBD-2 primers. RESULTS: PA induces time- and dose-dependently hBD-2 gene expression in nasal epithelial cells. Unstimulated epithelial nasal cells were able to express hBD-2 mRNA constitutively, whereas nasal fibroblasts showed no hBD-2 mRNA expression. Nasal polyps showed a comparable less hBD-2 gene expression then nasal turbinates. CONCLUSIONS: hBD-2 possibly mediates a specific, early starting antimicrobial defense strategy of the nasal mucosa. This hypothesis would explain persistent infections with PA through diminished hBD-2 gene expression.

Cells, Cultured↗
PubMed1986

In vitro secondary activation (memory effect) of avian vitellogenin II gene in isolated liver nuclei.

The vitellogenin II gene is specifically reactivated in vitro (secondary stimulation, memory effect) in purified liver nuclei that had ceased to express the gene in vivo a month after the roosters had received a single injection of estradiol (primary stimulation). The in vitro reactivation depends on the addition to the nuclei of nuclear and cytoplasmic extracts from estradiol-stimulated livers, polyamines (0.1-1.0 mM), and calmodulin (0.1 mM). Under identical incubation conditions the vitellogenin gene could not be reactivated in oviduct, embryonic, and immature chicken liver nuclei. Two other genes, those for ovalbumin and lysozyme, which are regulated by estradiol in the oviduct, could not be activated in the liver nuclei. The correct initiation of vitellogenin gene transcription in the liver nuclei was tested by primer extension studies. Addition of the antiestrogen tamoxifen (0.1 microM) to the system decreased vitellogenin mRNA synthesis by about 45% without affecting total RNA synthesis. Addition of quercetin (0.1 mM) and trans-flupenthixol (0.2 mM), inhibitors of nuclear protein kinase II and calmodulin-dependent kinase, respectively, inhibited the synthesis of vitellogenin mRNA by about 55% without affecting total RNA synthesis. The inhibitory effects of the antiestrogen and the kinase inhibitors were not additive, suggesting that both classes of inhibitor act on the same target or related targets. Depleting the estradiol receptors from the cell and nuclear extracts by means of estradiol-receptor antibodies covalently bound to Matrex beads reduced the stimulation of the vitellogenin gene by 40%. We conclude that in addition to the estradiol receptor and phosphorylation of nuclear protein(s) there are additional factors responsible for the in vitro secondary activation of the avian vitellogenin II gene.

Animals↗
PubMed1997

Rapamycin inhibits alpha 1-adrenergic receptor-stimulated cardiac myocyte hypertrophy but not activation of hypertrophy-associated genes. Evidence for involvement of p70 S6 kinase.

The 70-kD S6 kinase (p70S6K) has been implicated in the regulation of protein synthesis in many cell types and in the angiotensin II-stimulated hypertrophy of cardiac myocytes. Our purpose was to determine whether p70S6K plays a role in cardiomyocyte hypertrophy induced by the alpha 1-adrenergic receptor (alpha 1-AR) agonist phenylephrine (PE). PE stimulated the activity of p70S6K > 3-fold, and this increase was blocked by rapamycin, an immunosuppressant macrolide that selectively inhibits p70S6K. When administered for 3 days, PE stimulated a 30% increase in total protein content, a 2-fold increase in the incorporation of [14C]phenylalanine (14C-Phe) into protein, and a 50% increase in two-dimensional myocyte area. Rapamycin pretreatment (> or = 500 pg/mL) significantly inhibited each of these PE-stimulated changes. Two days of PE treatment resulted in a 1.6-fold increase in total RNA yield per dish, a 2-fold increase in incorporation of [14C]uridine into myocyte RNA, and increases in relative mRNA levels of the hypertrophy-associated atrial natriuretic factor (ANF, 2.1-fold) and skeletal alpha-actin (SK, 2.2-fold) genes. Although rapamycin abolished the PE-stimulated increases in total RNA and incorporation of [14C]uridine, it had no effect on the induction of the ANF and SK genes. LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K) activity, inhibited PE-stimulated increases in p70S6K activity and the incorporation of labeled precursors into myocyte protein and RNA. These results demonstrate that p70S6K is activated by the hypertrophic agent PE and that a PI3-K or PI3-K-like activity is required for p70S6K activation and myocyte hypertrophy. The data suggest that p70S6K activation may be required for PE-stimulated hypertrophy of cardiac myocytes. Our results demonstrate that intracellular signaling pathways responsible for transcriptional and translational responses diverge early after alpha 1-AR stimulation in cardiac myocytes.

Adrenergic alpha-1 Receptor Antagonists↗
PubMed1989

Androgen regulation of major mRNAs for proteins of the immature and adult mouse vas deferens.

When total RNA and poly (A)+ RNA extracted from the adult mouse vas deferens were translated in the rabbit reticulocyte lysate protein synthesizing system, they showed a similar electrophoretic pattern of about 20 protein bands of widely varying molecular weights. Within the total mRNA population, three functional mRNAs coding for proteins with molecular weights of 24, 34.5 and 36 kDa showed marked changes after 1 month of castration. The time-course of the response of the translatable proteins showed that while 3 days after castration the 24 and 36 kDa bands were missing, the mRNA coding for the 34.5 kDa band was markedly reduced only 20 days after castration. The negative effect of castration on the three mRNAs could be completely reversed by treatment with testosterone. When total RNA extracted from the immature mouse vas deferens was translated, the 24, 34.5 and 36 kDa protein bands were detectable in 10-day-old males and were synthesized in significant amounts between 10 and 20 days (24 and 36 kDa bands) or between 20 and 30 days (34.5 kDa band). Based on its electrophoretic and immunological properties, the 34.5 kDa protein band, which is predominant in the translation products, was identified as the major androgen-dependent protein previously described in vivo.

Age Factors↗
PubMed1976

Distinctive effects of mechlorethamine (NSC-762), cyclophosphamide (NSC-26271), and BCNU (NSC-409962) on transcription in Ehrlich cells.

Comparisons have been made between mechlorethamine, cyclophosphamide (CP), and BCNU as to the effects of chemotherapeutic drug levels on the transcription of RNA in vivo in Ehrlich cells. Mechlorethamine causes a dose-dependent depression in the synthesis of large Hn RNA transcripts in the nucleus and in the amount of large polysomal mRNA in the cytoplasm without apparent disturbance of polyadenylation and transport. Because of compensating increases in smaller Hn RNA and mRNA chains there is no inhibition of total RNA synthesis. CP seems to accelerate total Hn RNA and mRNA formation but inhibits polyadenylation. CP also produces an excess of short chains with only a minor depression of the synthesis of longer transcripts and messages. BCNU inhibits both RNA synthesis and polyadenylation but without disturbance of the size distribution of the polynucleotide chains. The results with mechlorethamine, in particular, are not easily explained on the basis of premature chain termination at the sites of DNA alkylation.

Adenosine Triphosphate↗
PubMedDate not supplied

Identification and localization of copper-zinc superoxide dismutase gene expression in rat testicular development.

Reactive oxygen species are highly toxic agents that appear to have an important role in male infertility. In order to understand the potential for the testis to be protected from reactive oxygen, the mRNA levels of the natural reactive oxygen scavenger, copper-zinc superoxide dismutase (SOD), were determined in testes and other organs in rats using northern analysis and in situ hybridization. Northern analysis of total RNA from organs of 60-day-old rats demonstrated an SOD mRNA with a transcript length of 0.77 kb; its concentration was highest in the kidney, liver, testis, and epididymis. In testis, northern analysis of total RNA demonstrated two mRNA transcripts of 0.77 kb and 0.94 kb. The concentrations of the 0.77-kb transcript varied only slightly between 10 and 100 days of age. In contrast, the 0.94-kb transcript became detectable by northern analysis between 30 and 40 days of age, then its concentration rose progressively to peak at 60 days. In situ hybridization studies demonstrated a uniform distribution of SOD mRNA within seminiferous tubules of prepubertal rats at 10 days of age and a heterogeneous, stage-specific pattern in older animals. In mature rats, the highest level of SOD mRNA was detected in tubules just prior to spermiation (stages VI-VIII). In conclusion, two SOD mRNA transcripts were identified in the rat testes that followed significantly different patterns of expression during development. In situ hybridization studies revealed that accumulation of the SOD mRNA in the seminiferous tubule was stage specific. These data suggest that SOD may play an important role during testicular development and spermatogenesis in rats.

Aging↗
PubMed1995

Determination of mRNA levels of cholesterol biosynthesis enzymes and LDL receptor using ribonuclease protection assay.

We designed a rapid method for determining mRNA content of cholesterol biosynthesis enzymes and LDL receptor (LDLR) using a ribonuclease protection assay (RPA). 32P-labeled cRNA fragments for genes of human LDLR and the enzymes HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), farnesyl pyrophosphate synthase (FPPS), and squalene synthase (SQS) were prepared by in vitro transcription. Total RNA prepared from HepG2 cells was hybridized with the cRNA probe and the hybridized mRNA was determined under protection from RNase digestion. Probe content used in this assay was excess in determining the desired mRNA in total RNA, and surplus probes were completely digested using RNase under standard conditions. When cells were cultured in DMEM supplemented with 10% fetal calf serum (FCS), mRNA levels of FPPS, SQS, and LDLR were about 4- to 7-fold higher than those of HMGS, HMGR, and MK. On incubation with DMEM supplemented with 10% lipoprotein-deficient serum (LPDS) for 8 h, all messenger RNA levels increased 1.5- to 3.5-fold. In addition, when the HMG-CoA reductase inhibitor compactin was added to 10% LPDS-DMEM, these levels increased even further and the change in mRNA level seemed to differ between the enzymes and LDLR. From these results, we conclude that RPA is a useful method for determining the very small amount of mRNA level of cholesterol biosynthesis enzymes and LDLR in the cell.

Base Sequence↗
PubMed1981

Selective inhibition of avian sarcoma virus protein synthesis in 3-deazaadenosine-treated infected chicken embryo fibroblasts.

The production of B77 avian sarcoma virions was inhibited more than 90% in infected chicken embryo fibroblasts that were treated with 100 microM 3-deazaadenosine, an inhibitor of adenosylhomocysteine hydrolase and, for this reason, an inhibitor of methylation reactions. This nucleoside analog at a concentration of 100 microM inhibited the rates of overall cellular protein synthesis and polyadenylated RNA synthesis by 40 to 50%. Rates of viral protein synthesis were compared, and the results indicated that in infected cells treated with 3-deazaadenosine syntheses of both the precursor of the gag proteins (pr76gag) and the precursor of the reverse transcriptase (pr180gag pol) were inhibited. Synthesis of the precursor of the viral envelope glycoproteins (pr92env) appeared to be affected less by the analog treatment. Most of the host polypeptides also continued to be synthesized in 3-deazaadenosine-treated cells. The fraction of the total RNA represented by virus-specific RNA in the 3-deazaadenosine-treated cells was approximately 40% of the fraction of the total RNA represented by viral RNA in control cells, as determined by hybridization kinetics. Therefore, there was a selective inhibition of viral RNA accumulation in the presence of 3-deazaadenosine. The amounts of genome-sized 35S and 38S RNAs were reduced compared with the amounts of 28S and 21S viral mRNA's. These results suggest that selective inhibition of the synthesis of viral proteins is due to selective decreases in the amounts of the mRNA's for these polypeptides.

Animals↗
PubMed1999

Nucleic acid-binding properties of low-density lipoproteins: LDL as a natural gene vector.

The role of apo B-100 as a transcription factor is indicated by the presence of regions in its primary structure that are similar to the DNA-binding domains of the transcription factors ISGF3gamma, STATs, IRFs, and SREBPs as well as by the presence of 11 RNA-binding KH domains. The Apo B-100 sequence also contains numerous bipartite nuclear localization sequences (NLS). A modified gel shift assay was used to show binding of highly purified preparations of human LDLs to fragmented genomic DNA, plasmid DNA, synthetic oligonucleotides (ISRE, 5'-GGGAAACCGAAACTG and E/C, E-box motif and CCAAT, adipocyte-specific genes promoter site), and total RNA from human liver. LDL was observed to bind preferentially to plasmid DNA containing the hCMV IE2 promoter region. In experiments using human liver total RNA, RNA for five different genes was recovered from LDL and VLDL bands. Gene transfection experiments using human skin fibroblast cells were used to study the gene transfer capacity of LDL. Cells transfected with a pEGFP-N1 plasmid DNA and LDL expressed functional GFP, as indicated by fluorescence, at approximately 3 hrs after transfection. Our results strongly support an alternative role for apo B-100, in toto or perhaps as functional fragments, in the control of gene expression and as gene transfer vector.

Amino Acid Motifs↗