Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The recent discovery of chemokine receptors as coreceptors for human immunodeficiency virus-type 1 (HIV-1) entry offers new avenues for investigating the pathogenesis of acquired immunodeficiency syndrome (AIDS)-related cytopenias. To this end, we sought to (1) phenotype human hematopoietic cells for CD4 and the HIV-1 coreceptors CXCR4, CCR5, CCR3, and CCR2b; (2) correlate CD4 and chemokine receptor expression with their susceptibility to HIV-1 infection; and (3) examine any potential interplay between inflammatory cytokines released during HIV-1 infection and regulation of chemokine receptor expression. Fluorescence-activated cell sorting (FACS) analysis of bone marrow mononuclear cells (BMMNC), cells derived from serum-free expanded hematopoietic lineages (colony-forming unit-granulocyte-macrophage [CFU-GM], colony-forming unit-megakaryocyte [CFU-Meg], and burst-forming unit-erythroid [BFU-E]), and CD34(+) cells showed differential expression of chemokine receptors and CD4 with some lineage specificity. Significantly, FACS-sorted CXCR4(+)/CD34(+) cells had the same clonogeneic potential as CXCR4(-)/CD34(+) cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of FACS-sorted human candidate stem cells (HSC; CD34(+), c-kit+, Rho123(low)) showed the presence of CXCR4 mRNA but not CD4 mRNA. Infection studies with HIV-1 Env-pseudotyped luciferase reporter viruses indicated that X4 Env (CXCR4-using) pseudotypes infected megakaryocytic cells, whereas R5 Env (CCR5-using) pseudotypes did not. Similarly, R5 but not X4 Env-pseudotyped viruses infected granulocyte-macrophage cells in a CD4/CCR5-dependent manner. Erythroid cells were resistant to R5 or X4 viral infection. Finally, we found that gamma-interferon treatment upregulated CXCR4 expression on primary hematopoietic cells. In summary, the delineation of chemokine receptor expression on primary hematopoietic cells is a first step towards dissecting the chemokine-chemokine receptor axes that may play a role in hematopoietic cell proliferation and homing. Furthermore, susceptibility of hematopoietic cells to HIV-1 infection is likely to be more complicated than the mere physical presence of CD4 and the cognate chemokine receptor. Lastly, our results suggest a potential interplay between gamma-interferon secretion and CXCR4 expression.
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PMID:Coreceptor/chemokine receptor expression on human hematopoietic cells: biological implications for human immunodeficiency virus-type 1 infection. 994 56

From July 1995 to December 1996, 3185 stool specimens from healthy children aged 6-59 months attending 6 dispensaries in the Antananarivo area were examined for poliovirus. The children had been routinely immunized according to the Expanded Programme on Immunization (EPI) schedule and received the last dose of oral polio vaccine (OPV) more than 1 month before stool collection. 99.4% of the children were immunized with at least 3 doses of OPV. HEp-2 cell culture revealed virus infections in 192 stools (6.0%), including 9 poliovirus (0.3%) and 183 nonpolio enterovirus isolates (5.7%). Infections occurred throughout the year, but incidence was higher during the hot and rainy season (P=0.01). Using a neutralization test with monoclonal antibodies and PCR-RFLP in two genomic regions coding for the VP1 capsid and RNA polymerase, 4 wild polioviruses (3 type 1 and 1 type 3) and 5 vaccine-related polioviruses (2 Sabin 1-like variants, 1 Sabin 2-like and 2 Sabin 3-like) strains were identified. The wild polioviruses were isolated at the beginning and the end of the dry season. Similar RFLP patterns were observed for the 3 wild type 1 polioviruses. Comparison of partial genomic sequences in the VP1/2 A region of 1 of the wild type 1 isolates with 2 wild type strains isolated in Antananarivo in 1992 and 1993 showed a divergence of at least 10% between the strains, suggesting at least two different pathways of transmission during this period. Our findings demonstrate that immunization with 3 doses of OPV did not prevent intestinal carriage of wild poliovirus strains, and that there is a risk of wild poliovirus transmission to susceptible children in the area. Multiple strategies are required to improve immunization coverage in Madagascar.
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PMID:Wild poliovirus circulation among healthy children immunized with oral polio vaccine in Antananarivo, Madagascar. 1020 74

Herpes simplex virus type 1 (HSV-1) infection alters the phosphorylation of the large subunit of RNA polymerase II (RNAP II), resulting in the depletion of the hypophosphorylated and hyperphosphorylated forms of this polypeptide (known as IIa and IIo, respectively) and induction of a novel, alternatively phosphorylated form (designated IIi). We previously showed that the HSV-1 immediate-early protein ICP22 is involved in this phenomenon, since induction of IIi and depletion of IIa are deficient in cells infected with 22/n199, an HSV-1 ICP22 nonsense mutant (S. A. Rice, M. C. Long, V. Lam, P. A. Schaffer, and C. A. Spencer, J. Virol. 69:5550-5559, 1995). However, depletion of IIo still occurs in 22/n199-infected cells. This suggests either that another viral gene product affects the RNAP II large subunit or that the truncated ICP22 polypeptide encoded by 22/n199 retains residual activity which leads to IIo depletion. To distinguish between these possibilities, we engineered an HSV-1 ICP22 null mutant, d22-lacZ, and compared it to 22/n199. The two mutants are indistinguishable in their effects on the RNAP II large subunit, suggesting that an additional viral gene product is involved in altering RNAP II. Two candidates are UL13, a protein kinase which has been implicated in ICP22 phosphorylation, and the virion host shutoff (Vhs) factor, the expression of which is positively regulated by ICP22 and UL13. To test whether UL13 is involved, a UL13-deficient viral mutant, d13-lacZ, was engineered. This mutant was defective in IIi induction and IIa depletion, displaying a phenotype very similar to that of d22-lacZ. In contrast, a Vhs mutant had effects that were indistinguishable from wild-type HSV-1. Therefore, UL13 but not the Vhs function plays a role in modifying the RNAP II large subunit. To study the potential role of UL13 in viral transcription, we carried out nuclear run-on transcription analyses in infected human embryonic lung cells. Infections with either UL13 or ICP22 mutants led to significantly reduced amounts of viral genome transcription at late times after infection. Together, our results suggest that ICP22 and UL13 are involved in a common pathway that alters RNAP II phosphorylation and that in some cell lines this change promotes viral late transcription.
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PMID:ICP22 and the UL13 protein kinase are both required for herpes simplex virus-induced modification of the large subunit of RNA polymerase II. 1036 8

A vaccinia virus-bacteriophage T7 RNA polymerase hybrid transient expression vector has been developed for complementation analysis of late gene functions in vaccinia virus. The conditionally defective virus ts21 was modified to express the bacteriophage T7 RNA polymerase. The derived virus, vtsT7, was conditionally defective in viral late gene expression but produced high levels of a target protein under the control of a T7 promoter at non-permissive temperatures. The level of beta-galactosidase expression under the control of a T7 promoter was slightly lower in vtsT7 infections than those with the prototypical T7 RNA polymerase vector vTF7.3. However, the levels of expression for the human immunodeficiency virus envelope gene, a protein which undergoes post-translational modification, was slightly higher in vtsT7 infections, suggesting that some proteins may be expressed better in the absence of vaccinia virus late gene expression. Infections using vtsT7 at a low m.o.i. at 39 degrees C resulted in the accumulation of high molecular mass, non-linear replicative intermediates of vaccinia virus DNA replication and high levels of expression of a transfected gene proximal to a T7 promoter. The virus vtsT7 provides a means for the analysis of potential trans-acting factors participating in vaccinia virus late processes such as resolution of DNA replicative intermediates.
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PMID:Vaccinia virus-bacteriophage T7 expression vector for complementation analysis of late gene processes. 1037 64

Reverse transcriptase (RT) isolated from Rous sarcoma virus (RSV) consists of heterodimeric RTalphabeta, RTalpha, and RTbeta. The alpha subunit (63 kDa) contains an N-terminal polymerase and a C-terminal RNase H domain. The N terminus of beta (95 kDa) corresponds to alpha with the integrase domain attached to the C terminus (32 kDa). We have constructed baculoviruses expressing the genes for alpha or beta or the entire pol (99 kDa). Infection of insect cells with recombinant virus yielded highly active and soluble RSV RT enzymes that could be purified to >90% homogeneity. HPLC gel filtration showed that alpha is a dimeric enzyme that can be partially monomerized upon the addition of 45% Me(2)SO. DNA synthesis on DNA-DNA and DNA-RNA primer-templates in the presence of competitor substrates revealed that alphabeta and beta as well as alpha are processive polymerases. However, the affinity of beta and alphabeta for primer-template substrates appears to be higher than that of alpha. All RSV enzymes investigated have the potential to displace RNA-RNA duplexes more efficiently than human immunodeficiency virus type 1 RT. Unlike human immunodeficiency virus type 1 RT, RSV RTs can catalyze an initial RNase H endonucleolytic cleavage of the RNA template but not a 3' --> 5' directed processing activity.
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PMID:Soluble Rous sarcoma virus reverse transcriptases alpha, alphabeta, and beta purified from insect cells are processive DNA polymerases that lack an RNase H 3' --> 5' directed processing activity. 1047 89

There are two types of infection caused by pathogenic microorganisms, intracellular infection and intercellular infection. Infection of pathogenic leptospira is an intercellular infection. The immunological reaction of host to intercellular infection is unique. The potential immunogen of an expressed protein should meet three criteria: it can be degraded (by antigen-present cells in the host); it should have antigenic epitope which can be recognized by specific antibodies and have at least one epitope that can be recognized by an MHC II protein and T cell receptor. In this study we report the cloning of an L. interrogans protein in plasmid rpDJt and the immunogencity of the expressed protein derivative. A genomic library of L. interrogans serovar lai strain 017 was constructed with the plasmid vector pUC18. Recombinant plasmids, designated pDJH2 and pDJ8 were screened from the bank. EcoRI-inserted fragment of 1. 9 kb recombinant DNA of pDJH2 was ligated into T7 RNA polymerase/promoter vectors (pT7-7). Then they were transformed into E. coli JM109 (De3), one of subclones, designated rpDJt was achieved. SDS-PAGE showed that the molecular weights of expression proteins were 68 kd and 23 kd respectively, designated p68 and p23. Purifying and isolating p68 and p23, we separated them from SDS-Polyacrylamide gels by using Side-Strip method. After fragmenting and electroeluting, p68 and p23 were injected into guinea pigs and rabbits. An extremely strong immune response to p68 was obtained since an anti-p68 antibody response could be detected to a dilution 1:524,288 (guinea pigs) and 1:262,144 (rabbits) by ELISA while anti-P23 antibody being 1:1024 (the same to guinea pigs and rabbits). The results of improved MTT and conA 3HTdR transformation methods showed the activities and proliferation of Th-cells were increased in guinea pigs after p68 immunization (IL-6, 83.25 IU/ml, IL-2, 28.75 IU/ml; RPI, 2.04, SI, 65.62%) Thlymphocyte existed in two subclasses, the Th1- and Th2-cells. A major role of Th2-cells is to "help" B-cells differentiate, replicate, and secrete antibody. The properties of these interactions explain why p68 makes good antigen and p23 does not. The antigens responsible for eliciting the production of protective antibodies are not known; however, several outer membrane proteins on L. interrogans are candidates for vaccine. Our results suggest that expresion protein p68 from recombinants (rpDJt) may be a candidate for gene engineered subunit vaccine for Leptospirosis.
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PMID:[Immunogenecity of expressed protein p68 from recombinant plasmid rpDJt in L. interrogans serovar lai]. 1068 17

Infection with high-risk human papillomaviruses (HPV), is the most significant risk factor for cervical cancer and it may be possible to prevent this malignancy by immunisation. Before immunisation programmes can be designed, however, it is necessary to know the age of acquisition and all routes of infection for these viruses. Sexual transmission is well documented and vertical transmission has also been demonstrated, although the frequency of transmission remains controversial. We previously showed that vertical transmission frequently results in persistent infection, and now present data on the prevalence of HPV-16 DNA (the most prevalent high-risk HPV type) in healthy children. Buccal samples from 267 healthy children aged 3-11 years were tested for HPV DNA by generic PCR (MY09/MY11), and a HPV-16 specific nested PCR. Reverse transcriptase (RT)-PCR was used to determine the prevalence of transcriptionally active HPV-16 infection in a subset of children. HPV-16 DNA was detected by nested PCR in 138 of 267 (51.7%) samples, whereas HPV DNA was detected in only 45 (16.8%) specimens by generic PCR, that has a lower analytical sensitivity. There were no significant differences in prevalence according to age or sex. Early region mRNA was detected by RT-PCR in six (11.3%) of 53 HPV-16 E5 DNA positive samples. HPV-16 E5 DNA sequences from 10 children confirmed the identity of the sequences detected and identified 13 HPV-16 variants.
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PMID:High prevalence of human papillomavirus type 16 infection among children. 1074 35

Infections with Haemonchus contortus are a major constraint on ruminant health world-wide. Young lambs are very sensitive to Haemonchus infection. Older lambs and sheep acquire immunity after a continuous or seasonal exposure to the parasite. The mechanisms underlying immunity are still not completely understood. Antibodies, in particular local IgA and IgE, certainly play a role. The role of IgG is less clear. Lymphocyte proliferation responses seem to correlate to immunity. Sheep that have high antigen-induced lymphocyte responses have a low susceptibility to infection. Furthermore, several studies have demonstrated that immunity against H. contortus is associated with mastocytosis and hyper-sensitivity reactions. More recently, increasing attention is being paid to the role of cytokines (interleukins and gamma-interferon) in the activation of specific defence mechanisms. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays to study cytokine mRNA expression have become available. The inability of young lambs to mount a significant Th2 response, which is normally characterized by high IgE levels, mastocytosis and eosinophilia, may account for the phenomenon of unresponsiveness in these animals.
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PMID:Immunological responses of sheep to Haemonchus contortus. 1087 10

The aim of this study was to generate urgently needed data on respiratory pathogens in German children using an economical and efficient tool. Nasopharyngeal aspirates of hospitalized children 0-16 years of age with an acute respiratory tract infection were tested by a nine-valent multiplex reverse-transcriptase polymerase chain reaction. Of 1281 children, 449 (35%) had an acute respiratory tract infection caused by at least one of the organisms studied; there were 29 cases of dual infection. At least 34-42% of severe acute respiratory tract infections in children under 5 years of age were caused by viruses. In children over 5 years of age, this proportion was 23% (P<0.001). Infection during the first 2 years of life was most frequently due to respiratory syncytial virus (n = 162 cases). Parainfluenza virus type 3 (n = 22) and type 1 (n = 14) were detected almost exclusively in children under 5 years of age. Influenza A (n = 90) and adenoviruses (n = 98) were prevalent in all age groups. The frequency of influenza B virus isolation (n = 17) rose significantly after the age of 5 years. Mycoplasma pneumoniae infection (n = 24 cases, 5.2%) was most frequent in 5- to 16-year-old patients. Only one case of Chlamydia pneumoniae infection was found. Since the distribution of pathogens within the different types of lower respiratory tract infections is very similar, it seems that host factors determine which form of lower respiratory tract infection develops in an individual patient. The multiplex reverse-transcriptase polymerase chain reaction may, in the future, become an important tool for epidemiological studies as well as for individual diagnosis.
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PMID:Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse-transcriptase polymerase chain reaction. 1089 33

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.
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PMID:Establishment of a rescue system for canine distemper virus. 1104 18


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