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Query: UNIPROT:P06889 (Mol)
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This study describes a nonisotopic polymerase chain reaction-reverse hybridization-based method (PCR-RH) for the one-step detection and genotyping of anogenital human papillomavirus (HPV) in a microwell format. HPV DNA was amplified and labeled by PCR using GP5+/GP6+ primers. Labeled amplicons were hybridized to 20 HPV type-specific capture probes anchored to the surface of plastic microwells and detected by an immunoenzymatic assay. Assay sensitivity was <50 pg labeled amplicon, and no cross-reactivity was observed, as determined by hybridizing serial dilutions of labeled PCR products to either matched or mismatched capture probes. The assay was tested on 66 clinical samples (23 specimens with normal histology, I fibropapilloma, 26 cervical intraepithelial neoplasia grade 1 [CIN1], 9 CIN2, and 7 CIN3) and compared with a method based on restriction fragment length polymorphism (RFLP) of PCR products. PCR-RH and PCR-RFLP performed equally well on clinical samples. The overall HPV detection rate was similar: 65.1% (43/66) for PCR-RH and 57.6% (38/66) for PCR-RFLP. HPV DNA was found in all CIN2 and CIN3 samples by both methods; however, PCR-RH detected more positives among normal biopsy samples and CINI cases. Overall, there was good agreement between the two genotyping methods, but RH yielded fewer cases with undetermined HPV genotype.
Diagn Mol Pathol 2001 Sep
PMID:One-step detection and genotyping of human papillomavirus in cervical samples by reverse hybridization. 1155 23

In previous studies, the HLA class II haplotype HLA DRB1*0401-DQB1*0301 was shown to correlate with susceptibility to HPV infection, CIN and cervical cancer while DRB1*0101-DQB1*0501 indicated protection. The present study was designed to identify naturally processed peptide sequences bound to the susceptibility and protective HLA DR-DQ molecules, and use this for T-helper epitope prediction from HPV 16. The HLA class II molecules were obtained by immuno-affinity purification of Epstein-Barr virus B lymphoblastoid cell lines (BCL) homozygous for HLA DQA1*0301-DQB1*0301 and HLA DQA1*0101-DQB1*0501. Peptide pools eluted from the HLA molecules were sequenced by Edman degradation. On the basis of the peptide sequence data obtained, the E6, E7, L1 and L2 proteins of HPV 16 were examined to identify sequences which are likely to bind to HLA DQB1*0301 and DQB1*0501. In addition, motif prediction as well as the binding affinity of predicted peptide motifs for HLA DRB1*0401 and DRB1*0101, the DR alleles associated with susceptibility and protection respectively, was accomplished using published data and a prediction algorithm for the naturally processed peptide sequences bound to these molecules. The HLA DQB1*0501 peptide ligand sequence showed that proline gives an outstanding signal at position 2, Asn/Arg at P1, aliphatic/aromatic amino acids in the central portion, a hydrophobic cluster at P5 with a small contribution by small polar residues and another cluster of aromatic residues towards the C-terminus. The HLA DQB1*0301 sequence also showed that proline gives an outstanding signal at position 2, Thr/Arg at P1, aliphatic/aromatic amino acids in the central portion and an aliphatic cluster with a small contribution by small polar residues at P5. There were no differences in the number of HPV peptides that were predicted as being capable of binding to HLA DQB1*0301 and HLA DQB1*0501, but more HPV peptide motifs were predicted to bind with high affinity to HLA DRB1*0101 than DRB1*0401. The results suggest that HPV 16 peptide epitopes bind with higher affinity to the protective than to susceptible HLA DR-DQ molecules which may lead to a more effective immune response.
Int J Mol Med 2001 Oct
PMID:Motif analysis of HLA class II molecules that determine the HPV associated risk of cervical carcinogenesis. 1156 79

The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) causes diseases ranging from mild, self-limiting pharyngitis to severe invasive infections. Regulation of the expression of GAS genes in response to specific environmental differences within the host is probably key in determining the course of the infectious process, however, little is known of global regulators of gene expression in GAS. Although secondary RNA polymerase sigma factors act as global regulators of gene expression in many other bacteria, none has yet been isolated from the GAS. The newly available GAS genome sequence indicates that the only candidate secondary sigma factor is encoded by two identical open reading frames (ORFS). These ORFS encode a protein that is 40% identical to the transcription factor ComX, believed to act as an RNA polymerase sigma factor in Streptococcus pneumoniae. To test whether the GAS ComX homologue functions as a sigma factor, we cloned and purified it from Escherichia coli. We found that in vitro, this GAS protein, which we call sigmaX, directed core RNA polymerase from Bacillus subtilis to transcribe from two GAS promoters that contain the cin-box region, required for transcription by S. pneumoniae ComX in vivo. On the other hand, GAS sigmaX did not promote transcription of a GAS promoter (hasA) expected to be dependent on sigmaA, the housekeeping or primary RNA polymerase sigma factor. Addition of monoclonal antibody that inhibited sigmaA-directed transcription had no effect on sigmaX-directed transcription, showing that the latter was not the result of contaminating sigmaA. Transcription of both cin-box-containing promoters initiated downstream of the cin-box and two different single basepair substitutions in the cin-box of the cinA promoter each caused a severe reduction of sigmaX-directed transcription in vitro. Thus, the cin-box is required for sigmaX-directed transcription.
Mol Microbiol 2001 Oct
PMID:A secondary RNA polymerase sigma factor from Streptococcus pyogenes. 1170 70

TA-HPV (therapeutic antigen-human papilloma virus) is a vaccine being developed by Xenova (formerly Cantab) for the potential treatment of cervical cancer. The antigen is intended to activate HPV-specific cytotoxic T-cells to attack tumor cells containing the viral antigen. Over 70% of patients with cervical cancer have tumor cells containing papillomavirus DNA [173070]. TA-HPV has reached phase IIa trials in 60 patients with high-grade anogenital intraepithelial neoplasia, including VIN3 (grade 3 vulvar intraepithelial neoplasia), to evaluate clinical efficacy [381386], [427159], [429895]. In addition, a phase II 'prime-boost' study of TA-HPV in combination with TA-CIN has been initiated at three centers across the UK [427159].
Curr Opin Mol Ther 2001 Dec
PMID:Technology evaluation: T-cell activator, Xenova. 1180 73

Human papillomavirus type 16 is a common sexually transmitted pathogen capable of giving rise to cervical intraepithelial neoplasia and invasive carcinoma through the expression and activity of two adjacent oncogenes: E6 and E7. Naturally occurring amino acid variation is commonly observed in the E6 protein but to a much lesser extent in E7. In order to investigate the evolutionary mechanisms involved in the generation and maintenance of this variation, we examine 42 distinct E6-E7 haplotypes using codon-based genealogical techniques. These techniques involve estimation of the ratio of nonsynonymous to synonymous substitutions (dn/ds) and allow testing for directional (positive) natural selection. Positive selection was detected for four codon sites within the E6 oncogene but not in any E7 codons. The amino acid compositions and locations of selected sites are described. Possible sources of natural selection including antiviral immune pressure and polymorphism of host cellular proteins are discussed.
J Mol Evol 2002 Oct
PMID:Evidence of diversifying selection in human papillomavirus type 16 E6 but not E7 oncogenes. 1235 68

The Papanicolaou smear has contributed to a decrease in cervical cancer rates in populations that receive regular screening. However, treatment of women with mildly abnormal cells is problematic because the majority of these women do not develop neoplasia. Thus, new techniques for identification of truly precancerous cells are needed. Characterization of cellular gene expression patterns is now possible through microarray techniques that survey the expression of large numbers of genes simultaneously. Here we have assessed the feasibility of combining new microscopic and molecular technologies to determine gene expression patterns in cervical intraepithelial neoplasia grade 3 cells recovered from liquid cytology-based Papanicolaou smear slides. Laser capture microdissection was used to retrieve cervical cells from ThinPrep prepared slides. The quality of RNA recovered from these cells proved suitable for reverse transcription polymerase chain reaction and for T7 RNA polymerase-based linear amplification of messenger RNA. We developed an optimized RNA amplification protocol that permitted microarray gene expression profiling in samples of as few as 20 cervical cells. This approach combining laser capture microdissection, linear RNA amplification, and microarray gene expression analysis will enable comparison of gene expression patterns between cytologically abnormal and normal cells taken from a single slide and may assist in the differential diagnosis of histologically difficult cases.
Appl Immunohistochem Mol Morphol 2003 Dec
PMID:Liquid-based pap smears as a source of RNA for gene expression analysis. 1466 62

The aims of the study were to investigate the relationship between human papillomavirus (HPV) DNA status and recurrence of cervical intraepithelial neoplasia (CIN) after loop excision (LEEP/LLETZ). Women (n=161) who underwent loop excision for CIN III and who were followed up prospectively for at least 4 years were the study cohort. Cervical smear cytology and testing for HPV DNA was performed at 3, 6 and 12 months prospectively and thereafter at intervals of 6-12 months, using the PCR method with a consensus primer targeting the L1 region. There has been no recurrence in 141 (81.6%) out of 161 subjects, while squamous intra-epithelial lesions (SIL) of low or high grade on cytology and CIN grade I-III on histology have been detected in 20 subjects. Prior to loop excision, HPV DNA was detected in 17 subjects who developed recurrence (9 had type 16, 2 each had types 18 and 52, and 1 each had types 31, 51, 58, and unknown). Within 3 months postoperatively, 12 (70.7%) subjects became negative for HPV, but 2 remained positive for the same type (1 each had types 16, 18), along with high-grade SIL on cytology, and CIN III on histology within 6 months, so repeat loop excision was performed. On the other hand, cytological findings were normalized in all transiently infected subjects within 18-36 months. Our results suggest that loop excision has improved HPV infection in many cases of CIN III and the persistent infection with a high-risk type of HPV is a predictor of the recurrence of CIN grade III.
Int J Mol Med 2004 Apr
PMID:Human papillomavirus DNA status after loop excision for cervical intraepithelial neoplasia grade III - A prospective study. 1501 Aug 61

Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.
J Mol Diagn 2004 May
PMID:Performance assessment of eight high-throughput PCR assays for viral load quantitation of oncogenic HPV types. 1509 67

Integration of the human papillomavirus (HPV) genome is thought to be one of the causes of cancer progression. However, there is controversy concerning the physical status of HPV 16 in premalignant cervical lesions, and there have been no reports on the concordance between detection of the integrated form of HPV16 by real-time PCR and by in situ hybridization. We investigated specimens of cervical intraepithelial neoplasia (CIN) and invasive carcinomas for the physical status of HPV 16 by real-time PCR and in situ hybridization. The presence of the integrated form was detected by both real-time PCR and in situ hybridization in zero of four cases of CIN1, three of six cases of CIN2, nine of 27 cases of CIN3, and two of six cases of invasive carcinomas. Integrated HPV 16 was present in some premalignant lesions but was not always present in carcinomas. The concordance rate between the two methods for the detection of the presence of the integrated form was 37 of 43 (86%) cases. Real-time PCR and in situ hybridization were found to be complementary and convenient techniques for determining the physical status of the HPV genome. We conclude that a combination of both methods is a more reliable means of assessing the physical status of the HPV genome in cervical neoplasia.
Diagn Mol Pathol 2005 Jun
PMID:Comparison between in situ hybridization and real-time PCR technique as a means of detecting the integrated form of human papillomavirus 16 in cervical neoplasia. 1590 94

A wide interobserver variation is seen even among competent histopathologists in the routine diagnosis of cervical intraepithelial neoplasia (CIN). As a result, early detection of low-grade CIN (CIN 1) lesions, in particular, remains a major challenge both in routine diagnosis and in cervical screening. In this chapter, the salient diagnostic features of human papillomavirus infection and CIN lesions are demonstrated.
Methods Mol Med 2005
PMID:Histological analysis of cervical intraepithelial neoplasia. 1635 Mar 95


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