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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A rapid and sensitive one-step polymerase chain reaction (PCR) assay was developed for use in identifying type I and type II herpes simplex virus (HSV). Although the nucleotide sequences of the two HSV subtypes are quite similar, common and type-specific sequences 20 nucleotides in length could be deduced in the thymidine kinase gene. Oligonucleotide primers targeted to the type-specific regions generated products of different sizes that served to distinguish two HSV types. Type-specific PCR amplification products were verified by restriction enzyme digestion. Specificity of the HSV PCR was established by the lack of amplification of other herpes-group viruses including cytomegalovirus, Epstein-Barr virus, and varicella zoster virus. Extraction of DNA from clinical materials (throat swabs, vesicular swabs, cerebrospinal fluid and eye discharge) yielded an amplification product of the predicted size for each HSV type. Thus, this PCR system provides a rapid, sensitive and specific assay that can supplement the currently available modalities for detecting and typing HSV.
Mol Cell Probes 1994 Jun
PMID:One-step determination of herpes simplex virus types I and II by polymerase chain reaction. 796 91

The possibility of detecting cytomegalovirus (CMV) in formalin-fixed tissues by polymerase chain reaction (PCR) was evaluated in necroposies from lung tissues in a total of 24 patients who either had received organ transplants or were immunocompromised. PCR using two different pairs of primers for amplification of the major immediate early antigen of CMV was performed on fresh tissues and tissues fixed for 24, 48, and 72 h in neutral buffered formalin and compared to immunohistochemistry (IHC) and in situ hybridization (ISH). The fresh tissues of nine patients with serological evidence for acute CMV infection were all positive for CMV by PCR. After formalin fixation, the majority of the patients failed to show distinct signals with one or both pairs of primers as measured by densitometry. In contrast to this, fresh tissues of 15 patients without signs of an acute CMV infection were found either negative or weakly positive by PCR. Using IHC or ISH, positive results were observed only in five of nine and four of nine patients with acute CMV infection, respectively. These data demonstrate that, if only formalin-fixed tissue is available, PCR for CMV detection should be performed using two pairs of primers and should be supported by IHC.
Diagn Mol Pathol 1994 Sep
PMID:Influence of formalin fixation on the detection of cytomegalovirus by polymerase chain reaction in immunocompromised patients and correlation to in situ hybridization, immunohistochemistry, and serological data. 798 90

In situ hybridization (ISH) provides a means for identifying viral genomes in the context of tissue pathology. We have developed a specific and sensitive ISH probe for the detection of cytomegalovirus (CMV) DNA in formalin-fixed, paraffin-embedded tissue sections. Digoxigenin-11-dUTP was incorporated into a 435-base pair fragment of the CMV Major Immediate Early (MIE) gene with use of the polymerase chain reaction (PCR). Hybridized probe was detected by reaction with antidigoxigenin antibody coupled to alkaline phosphatase and chromogenic substrates. This method has detected CMV infection in routine clinical specimens from a variety of tissue types, including colon, kidney, liver, and stomach. Infection in cells with and without characteristic inclusions is revealed with this probe. The background is so low that single infected cells are detected unambiguously. No cross-hybridization was observed with cells infected with other viruses of Herpesviridae. This approach may be useful for producing probes for the detection of other viral genomes in tissue sections.
Diagn Mol Pathol 1994 Sep
PMID:PCR production of a digoxigenin-labeled probe for the detection of human cytomegalovirus in tissue sections. 798 96

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.
Mol Cell Biol 1994 Jul
PMID:Multiple instability-regulating sites in the 3' untranslated region of the urokinase-type plasminogen activator mRNA. 800 88

Extracellular matrix (ECM)-cell interactions are essential for the regulation of many genes in differentiated cell types. A number of expression vectors that work well in cells cultured on tissue-culture plastic appear to be inactive or sporadically active in vivo. We reasoned that these responses also may be influenced by the ECM. We therefore examined three commonly used viral enhancers and found that they all responded either positively or negatively to the presence of exogenous ECM. Using mouse mammary epithelial cells, we found that a mouse mammary tumor virus enhancer linked to its own promoter or to a truncated (and by itself inactive) beta-casein promoter drove transcription efficiently only when the cells were in contact with an ECM (more than a 100-fold induction over tissue-culture plastic). Similarly, the cytomegalovirus enhancer was more active in cells in contact with ECM. In contrast, the simian virus 40 enhancer, linked to the beta-casein promoter, was 12-fold more active in cells on tissue-culture plastic. This activity was strongly reduced when the cells interacted with ECM. Thus, we conclude that different enhancers can respond to ECM by either activating or suppressing transcription. This observation has important implications for understanding the mechanisms of promoter action and for designing expression systems for use in gene therapy.
Mol Carcinog 1994 Jun
PMID:Transcriptional activation by viral enhancers: critical dependence on extracellular matrix-cell interactions in mammary epithelial cells. 803 66

A systematic analysis of DNA polymerase of human herpes simplex type 1 virus, cytomegalovirus, and human type 2 adenovirus with the help of a broad set of modified substrates of these enzymes has been carried out. It revealed compounds capable of inhibiting the DNA synthesis catalyzed both by all three enzymes and DNA polymerase alpha from human placenta. Compounds have been found which effectively and specifically inhibit the DNA synthesis catalyzed by some of the abovementioned enzymes. It has been shown that the molecular mechanism of inhibition consists either in the termination of DNA elongation or in inhibition without incorporation into the growing DNA chain.
Mol Biol (Mosk)
PMID:[Inhibitory analysis of DNA polymerases from human viruses using modified substrates]. 805 45

Previous serological analysis of untreated splenocytes and L cell transfectants expressing the wild-derived mouse major histocompatibility complex (MHC) class I molecule, Lw16, demonstrated the presence of two forms of this molecule in the cell lysates, one reactive with both the alpha 2 domain-reactive monoclonal antibody (mAb) 30-5-7 and the alpha 3 domain-reactive mAb 28-14-8 (30-5-7+ 28-14-8+), and the other reactive with only the latter of the two (30-5-7- 28-14-8+). Furthermore, the analysis suggested the presence of both forms on the cell surface. Due to the similarity of Lw16 to the inbred mouse-derived Ld molecule, we tested a panel of Ld-restricted and control peptides for their ability to bind to Lw16 molecules. Here, we report that two Ld-restricted viral peptides, lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) 118-126 and murine cytomegalovirus (MCMV) pp89 168-176, significantly increase the number of Lw16 molecules on the cell surface as measured by the mAb 28-14-8, and the proportion of those molecules that are recognized by the mAb 30-5-7. This was further supported by an increase in mAb 30-5-7-reactive molecules in L.Lw16 cell lysates following treatment with either of these peptides. Examination of the stability of the different forms on the cell surface suggested that the 30-5-7+ Lw16 molecules induced with these peptides were unstable and probably lost their Ld-restricted peptides to generate 30-5-7- 28-14-8+ molecules; these latter molecules were also unstable. In contrast, putative 30-5-7+ and 30-5-7- 28-14-8+ Lw16 molecules on untreated cells were stable. Together, these results suggest that two Ld-restricted, viral peptides can induce assembly of or stabilize 30-5-7+ 28-14-8+ Lw16 molecules, mimicking endogenous self peptides. However, the association of the Ld-restricted peptides with Lw16 is apparently not optimal, since it results in unstable Lw16 molecules.
Mol Immunol 1994 Aug
PMID:Association of Ld-restricted peptides with the wild-derived mouse Lw16 MHC class I molecule. 806 77

The major immediate early gene of human cytomegalovirus has been cloned. The fragment of the immediate early protein containing 351 amino acids has been fused with cro-beta-galactosidase and expressed in Escherichia coli cells. The obtained recombinant protein reacts in immunoblotting with the antibodies in human sera from patients suffering from acute cytomegalovirus infection.
Mol Gen Mikrobiol Virusol
PMID:[Cloning the human cytomegalovirus immediate-early gene and expression of it in Escherichia coli]. 806 79

The transfer of exogenous DNA in fish represents a powerful strategy to study the regulation of gene expression in vivo. The African catfish (Clarias gariepinus) was chosen for this study because of its scientific and economic importance due to its easy husbandry, its short developmental period, and its value as a protein source in Africa and Asia. Fertilized eggs (1- and 2-cell stage) were cytoplasmatically injected with either supercoiled or linearized plasmids harboring the fusion genes encoding beta-galactosidase (lacZ) or luciferase (Luc) without a promoter or fused to the promoter/enhancer of human cytomegalovirus (CMV). Replication of the exogenous DNA peaked at 4 hours (early gastrula) and again at 2 days (which corresponds to the developmental stage of yolksac resorption). Foreign DNA persisted during embryogenesis, and it was still detectable 8 months after injection. In vivo transient expression of both CMV fusion genes was mosaic and peaked within 24 hours after DNA injection. Transient expression of the luciferase reporter gene could be detected with a much higher sensitivity than the lacZ gene. These data establish African catfish as a suitable in vivo assay system, and they confirm the luciferase reporter gene as a high quality reporter gene in fish.
Mol Mar Biol Biotechnol 1994 Apr
PMID:Replication, expression, and fate of foreign DNA during embryonic and larval development of the African catfish (Clarias gariepinus). 808 84

Selective expression of subtypes of receptors in mammalian cell lines permits the study of the regulation of receptors in a homogeneous population of cells growing under controlled conditions. cDNAs encoding the human D2L and D2S receptors were ligated into a eukaryotic expression vector, pRc/CMV. The resulting plasmid, which contains a cytomegalovirus promoter for high expression levels, was used for stable transfection of 293 cells, a human kidney cell line. Expression of D2L and D2S receptors in 293 cells was confirmed by radioligand binding assays with [125I]NCQ 298. The pharmacological properties of the expressed receptors were comparable to those of receptors in rat striatal homogenates and in other transfected cell lines. D2L and D2S receptors were coupled to inhibition of cAMP accumulation in 293 cells. Incubation of 293-D2L cells with agonists resulted in an increase in the density of D2 receptors without a change in the affinity of the receptors for [125I]NCQ 298. This effect was time dependent, with a t1/2 of approximately 6 hr. The dose dependence of up-regulation followed the pharmacological profile expected of a D2 receptor, with an order of potency of N-propylnorapomorphine (NPA) > quinpirole > dopamine. The density of receptors was further increased by incubation of cells with agonist together with forskolin or 8-bromo-cAMP. D2S receptors responded similarly to D2L receptors to treatment with NPA and forskolin. Exposure of 293-D2L cells to the beta-adrenergic receptor agonist isoproterenol did not change the density of D2L receptors. Similarly, NPA had no effect on levels of endogenously expressed beta-adrenergic receptors in 293-D2L cells, as assayed by binding of [125I]iodocyanopindolol. Levels of beta-adrenergic receptors in transfected 293-beta 2 or 293-D2L cells did not increase after exposure to NPA but decreased after exposure to isoproterenol. Cells expressing D2L receptors were incubated with antagonists, including SCH-23390, sulpiride, haloperidol, clozapine, and epidepride, alone or in combination with NPA. Incubation of cells with SCH-23390 had no effect on the density of D2 receptors, and SCH-23390 did not block the effect of NPA. D2-selective antagonists increased the density of receptors. D2L receptor mRNA levels were unchanged during agonist treatment. This suggests a role for translational or post-translational mechanisms in the regulation of D2 receptor levels in transfected cell lines.
Mol Pharmacol 1993 Aug
PMID:Paradoxical regulation of dopamine receptors in transfected 293 cells. 810 83


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