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R D Burk

Publications and source records attributed to R D Burk.

At least 127 records · Page 7Linked to original sources
PubMed1989

Molecular diagnosis of genital human papillomavirus infection: comparison of two methods used to collect exfoliated cervical cells.

Human papillomavirus infection is implicated as an etiologic agent in the development of neoplasia and invasive carcinoma of the cervix. To detect human papillomavirus infection of the cervix, cells must be collected and assayed for human papillomavirus-related deoxyribonucleic acid sequences. Gynecologists and other clinical investigators generally use an exocervical spatula scrape and an endocervical swab for cell collection, analogous to Papanicolaou smear collection. However, inadequate cell recovery is common. To overcome this problem, we have developed the cervicovaginal lavage method for human papillomavirus detection. In the present study we compared the cervicovaginal lavage method with the widely used scrape-swab method in 48 women referred for colposcopic examination. After a Papanicolaou test, two samples were obtained from each woman, either with cervicovaginal lavage followed by scrape-swab or with the scrape-swab followed by cervicovaginal lavage. Human papillomavirus types were assessed by restriction analysis and Southern blot hybridization. In 21 women (44%) test results were positive for human papillomavirus with both the scrape-swab and cervicovaginal lavage cell collection methods; in nine women (19%) test results were positive only with the cervicovaginal lavage method; and in 18 women (38%) results were negative for human papillomavirus with both techniques. None of the women had human papillomavirus detected by scrape-swab without also having it detected with cervicovaginal lavage. The human papillomavirus deoxyribonucleic acid types identified were concordant in the 21 women whose infections were detected with both sampling methods, although the second virus type was detected only with cervicovaginal lavage in one woman who had a mixed genital tract infection. We concluded that cervicovaginal lavage is a more sensitive cell collection method than the scrape-swab technique for assessing human papillomavirus infection of the cervix.

Blotting, Southern↗
PubMed1989

Comparison of Cytobrush and cervicovaginal lavage sampling methods for the detection of genital human papillomavirus.

The development of an accurate method for the detection and typing of genital human papillomavirus is of substantial clinical importance. This virus has been implicated as an etiologic agent in the development of cervical neoplasia. To detect human papillomavirus infection with maximum sensitivity, cells must be collected and assayed for human papillomavirus deoxyribonucleic acid. We compared two noninvasive methods of sampling exfoliated cervical cells--cervicovaginal lavage and scrape-Cytobrush. Seventy-four patients newly referred to the colposcopy clinic were divided randomly for cell sampling by either cervicovaginal lavage followed by scrape-Cytobrush or, conversely, scrape-Cytobrush followed by cervicovaginal lavage. Restriction analysis and Southern blot hybridization were used to test all the samples thus obtained for human papillomavirus. Overall, test results from 42 patients (56.8%) were positive for human papillomavirus deoxyribonucleic acid. Twenty-six (31.1%) tested positive for human papillomavirus by both sampling methods, and 32 (43.2%) tested negative for human papillomavirus by both methods. One (1.4%) tested positive with scrape-Cytobrush sampling but negative with cervicovaginal lavage, while 15 (20.3%) tested negative with scrape-Cytobrush but positive with cervicovaginal lavage (p less than 0.001, McNemar's test). These data, combined with previous work from our group, suggest that, of the available methods, cervicovaginal lavage, coupled with human papillomavirus deoxyribonucleic acid hybridization, is the most sensitive noninvasive method for harvesting cells for molecular identification of human papillomavirus in the female lower genital tract.

Blotting, Southern↗
PubMed1989

DNase I hypersensitive site maps to the HBV enhancer.

Two lines of HBV transgenic mice (derived from G7 and G26) have been produced, each of which contains a unique locus of HBV DNA and expresses 2.1-kb HBsAg transcripts preferentially in liver and kidney tissues. To investigate the regulation of HBV expression in these mice, we have examined the state of methylation and the chromatin structure in and around the HBV sequences in tissues with and without HBV gene expression. Hypomethylation of HpaII and HhaI sites in and around the HBV sequences strongly correlated with HBV gene expression, although it was clearly not sufficient for HBV expression. Alterations in chromatin configuration were detected by DNase I digestion which identified a major hypersensitive site (HS) in liver and kidney tissue. By restriction enzyme mapping and indirect end-labeling, the HS was localized to the region of the HBV enhancer in both lines of HBV transgenics. The presence of this DNase I hypersensitive site was necessary but not sufficient for HBV expression, since it was also detected in tissues not expressing HBV. An additional DNase I hypersensitive site was mapped to the core promoter region of the G7 transgene in liver and kidney tissue but not in G26 tissues. The identification of a DNase I hypersensitive site mapping to the HBV enhancer region supports the notion that this region can interact with cellular proteins and is involved in the regulation of viral gene transcription in vivo.

Animals↗
PubMed1989

Developmental regulation of hepatitis B surface antigen expression in two lines of hepatitis B virus transgenic mice.

Two lines of hepatitis B virus (HBV) transgenic mice, designated G7 and G26, show preferential expression of the 2.1-kilobase hepatitis B surface antigen (HBsAg) RNA transcript in liver and kidney tissues (R. D. Burk, J. A. DeLoia, M. K. ElAwady, and J. D. Gearhart, J. Virol 62:649-654, 1988). This transcript was first identified in transgenic mice at gestational day 14 and was detected at similar or increased levels through birth and early development. However, in contrast to 2.1-kilobase HBsAg mRNA levels, HBsAg protein levels in serum decreased shortly after birth. Thereafter, serum HBsAg increased 100-fold to adult levels, with a corresponding 5- to 10-fold increase in HBsAg mRNA levels. In addition, adult males have higher levels of HBsAg in serum than females. HBsAg in serum in males was reduced approximately 50% by surgical castration and was restored to near-normal levels by testosterone supplementation. Since both transgenic lines show similar patterns of gene expression, we suggest that HBsAg gene expression is determined by viral regulatory elements in response to host factors. Whether tissue specificity, developmental regulation, and sexual dimorphism of expression of the exogenous HBV sequences were determined by single or multiple HBV regulatory elements remains to be determined.

Age Factors↗
PubMed1988

Detection of human papillomavirus deoxyribonucleic acid in exfoliated cervicovaginal cells as a predictor of cervical neoplasia in a high-risk population.

Specific types of human papillomavirus are currently implicated as etiologic agents of precancerous and cancerous lesions of the cervix. We have previously described the use of cervicovaginal lavage and molecular hybridization to detect human papillomavirus infections of the cervix. We report here the predictive value of this method of human papillomavirus detection to identify women with biopsy proved dysplastic and cancerous lesions of the cervix. One hundred ninety-one women from a city hospital colposcopy clinic underwent concurrent Papanicolaou smear, cervicovaginal lavage, and coloposcopically directed cervical biopsy. Human papillomavirus deoxyribonucleic acid was detected in 114 (59.7%) of these women. Of the positive results, human papillomavirus type 16 accounted for 23.7%, human papillomavirus type 18 for 10.5%, human papillomavirus type six or 11 for 6.2%, related human papillomavirus types for 52.6%, and 7.0% contained more than one type. The distribution of human papillomavirus types was similar in both women younger than 40 years of age and in older women. Eighty-nine of 128 (69.5%) women less than 40 years old with cervical lesions had positive findings of human papillomavirus, and 18 of 29 (62.1%) older women with cervical lesions had positive findings of human papillomavirus. Detection of human papillomavirus types 16 and 18 identified only 35 of 157 (22.3%) women with cervical lesions. The sensitivity of detecting all types of human papillomavirus as a predictor of a biopsy proved lesion (68.0%) was comparable with the sensitivity of cytologic examination alone (74.0%). However, human papillomavirus detection combined with the Papanicolaou smear provided an increased overall sensitivity of 89.3% (p less than 0.01). In fact, women either positive for human papillomavirus or having abnormal cytologic findings were 11.8 times more likely to have a biopsy proved cervical lesion than human papillomavirus-negative women with negative cytologic results (95% confidence interval for odds ratio: 5.3 to 26.6). We conclude that the sensitivity of cytologic examination plus human papillomavirus detection is superior to the use of either cytologic studies or human papillomavirus detection alone in identifying patients with cervical lesions.

Adolescent↗
PubMed1988

Interrupted replication of hepatitis B virus in liver tissue of HBsAg carriers with hepatocellular carcinoma.

To search for events underlying reduction of peripheral viremia and integration of hepatitis B virus (HBV) DNA into the liver cell genome in long-term virus carriers with hepatocellular carcinoma, paired samples of liver and tumor tissue were analyzed by molecular hybridization and immunological methods. Most tumor tissues contained integrated viral DNA; in none was extrachromosomal HBV DNA detected. Integrated HBV DNS was also found in peritumor liver tissue in the majority of patients. However, liver of patients either with or without peripheral viremia also contained free HBV DNA and replicative intermediates. In three nonviremic patients with replicative HBV DNA in liver, viral core antigen expression was markedly reduced or absent, whereas viral envelope protein (surface antigen) expression was normal. In one case, replicative intermediates in liver were sensitive to DNase I digestion, indicating that viral DNA was not encapsidated in normal viral core particles. These results suggest that decreased or defective core antigen production can lead to reduced viremia associated with blocked virus assembly/secretion and accumulation of unencapsidated HBV DNA replicative intermediates in the liver cell. Accumulation of such HBV DNA molecular forms in the liver may lead to an increased propensity for HBV DNA to integrate into the host genome, which has been found with high frequency in hepatic neoplasms from patients infected with hepatitis B virus.

Carcinoma, Hepatocellular↗
PubMed1988

Tissue preferential expression of the hepatitis B virus (HBV) surface antigen gene in two lines of HBV transgenic mice.

Two transgenic mice were produced by microinjection of the entire hepatitis B virus (HBV) genome as a 3.2-kilobase EcoRI DNA fragment into one-cell embryos. Each animal contained a single, unique locus of HBV sequence. One founder animal, G7, contained a partially deleted HBV genome lacking both putative HBV surface antigen (HBsAg) promoters. The other animal, G26, contained greater-than-genome-length HBV sequences organized as a partial head-to-tail dimer. Both transgenic animals transmitted the HBV sequences in a Mendelian fashion, and all subsequent transgenic animals had detectable HBsAg in the serum. Expression of HBV sequences in tissues from G7- and G26-derived mice showed preferential expression of the 2.1-kilobase HBsAg RNA transcript in liver and kidney tissues by Northern (RNA) blot analysis. These data are consistent with the notion that HBV DNA contains cis-acting regulatory sequences which are responsible for the predominant expression of HBsAg transcripts in the liver and kidney of transgenic mice.

Animals↗
PubMed1988

Head and neck squamous cell carcinomas associated with human papillomaviruses and an increased incidence of cervical pathology.

Human papillomaviruses (HPVs) have been identified in benign and cancerous epithelial lesions of the female genital tract. They have also been identified in papillomata and cancers of the upper aerodigestive tract. This study investigates the hypothesis that lesions of the cervicovaginal area are more common in women with cancers of the head and neck region. The presence of HPV in lesions of both regions is examined. Seven female patients with cancer of the upper aerodigestive tract had DNA analysis of their carcinoma specimens. HPV type 16 was found in two of the seven (28%). Fourteen female patients with upper aerodigestive tract cancers had Papanicolaou smears to search for cytologic evidence of HPV infection, and cervicovaginal lavages to analyze DNA from exfoliated cervical cells. Five of thirteen (38%) Papanicolaou smears revealed koilocytotic atypia and three of these patients had HPV DNA types 16 or 18 identified in the cervical lavage. The incidence of cervical atypia noted is 13-fold greater than average. One patient had HPV type 16 in both her supraglottic cancer and in her cervicovaginal lavage. Evidence of HPV infection at two separate anatomic sites suggests a systemic susceptibility to HPV infection.

Carcinoma, Squamous Cell↗
PubMed1988

The glucocorticoid receptor recognizes a specific nucleotide sequence in hepatitis B virus DNA causing increased activity of the HBV enhancer.

The hepatitis B virus (HBV) genome contains a specific DNA binding site for the glucocorticoid receptor. Using DNase I footprinting, this binding site was localized at HBV map positions 341-370 clockwise from the EcoRI site. The DNA sequence protected in the footprint contains two tandem copies of the GRE core hexanucleotide 5'-TGTTCCT-3'. Deletion analysis and reconstruction experiments in plasmid expression vectors demonstrated that this glucocorticoid receptor binding sequence serves as a signal for augmenting glucocorticoid-dependent activity of the HBV enhancer, which is located approximately 730 nucleotides downstream in the HBV genome. Even though it does not serve as an independent enhancer element, the HBV glucocorticoid receptor domain can therefore be categorized as a functional GRE.

Base Sequence↗
PubMed1987

Molecular analysis of integrated human papillomavirus 16 sequences in the cervical cancer cell line SiHa.

Human papillomavirus (HPV) 16 is frequently found integrated into cervical cancer cell genomes and these integrations are thought to play a role in tumorigenesis. To investigate the mechanisms of HPV integration and its effect on transcription and chromosomal sequence organization, we have cloned and analyzed the HPV16 integration from the cervical cancer cell line SiHa. Restriction analyses and Southern blotting indicated that approximately 95% of an HPV16 genome was integrated without gross rearrangement. Sequence analysis of the cellular-viral DNA junctions revealed that integration had occurred within the E2 and E4 ORFs where 251 bp of viral sequence was deleted. One viral terminus occurred within sequences of an Alu repeat and a 4-bp homology was present at the site of recombination. Using unique cellular flanking DNA probes, a 4.8-kb deletion of cellular sequences was detected at the site of viral integration. The chromosomal location of the viral integration and cellular deletion were mapped to chromosome 13 using a rodent X human somatic cell hybrid panel. Northern blot analysis using viral subgenomic and 3' cellular probes revealed transcription from the 3' portion of integrated HPV16 (E6, E7, E1) and flanking cellular sequences. The observation of viral-cell transcripts and chromosomal deletions associated with HPV integration may indicate that such events are part of a multistep mechanism leading to the development of cervical cancer.

Base Sequence↗
PubMed1986

Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: correlation with biopsy results and Papanicolaou smear.

Human papillomaviruses have previously been identified by molecular hybridization in the majority of dysplastic and cancerous lesions of the cervix. Since human papillomavirus types 16 and 18 have been strongly associated with cervical cancer, the identification of patients infected with these specific human papillomavirus types may provide useful prognostic information. We have developed a painless, noninvasive cervicovaginal lavage technique to collect exfoliated cervicovaginal cells, which can be reliably analyzed for the presence of human papillomavirus deoxyribonucleic acid by Southern blot analysis with the use of deoxyribonucleic acid cloned from human papillomaviruses 6, 11, 16, and 18. In a prospective study of 60 women referred to a colposcopy clinic for evaluation of abnormal Papanicolaou smears, we have detected human papillomavirus deoxyribonucleic acid in 16 of 17 (94%) women with a Class III (dysplasia) or IV (carcinoma in situ) Papanicolaou smear, five of 11 (45%) women with a Class II (atypical) Papanicolaou smear, and 10 of 34 (29%) women with a normal Papanicolaou smear. Detection of human papillomavirus deoxyribonucleic acid in cervicovaginal cells was indicative of a dysplastic cervical lesion in 19 of 20 (95%) patients irrespective of Papanicolaou smear results. We conclude that human papillomavirus deoxyribonucleic acid analysis in cervicovaginal cells is a sensitive method to detect dysplastic lesions of the cervix and may be useful in identifying patients with specific types of human papillomavirus infection, who are at risk to develop cervical cancer.

Biopsy↗
PubMed1986

Human papillomaviruses of different types in precancerous lesions of the uterine cervix: histologic, immunocytochemical and ultrastructural studies.

In a prospective study of 34 women with abnormal Papanicolaou smears, biopsy and cervicovaginal lavage specimens were analyzed for the presence of human papillomaviruses (HPVs) by Southern blot analysis and probes for HPVs 6, 11, 16, and 18. In 22 of the 23 patients with cervical lesions (96%), HPV DNA was identified in one or more specimens. All patients in whom HPV DNA was found had either koilocytotic or dysplastic lesions on biopsy or Papanicolaou smear. Immunocytochemical demonstration of HPV in biopsy samples was associated with the presence of large amounts of HPV DNA and with the ultrastructural identification of viral particles. The presence of HPV DNA in cervical biopsy specimens was limited to discrete geographic areas of the cervix with histologic abnormalities. Although HPV 16 and other related HPV types were found in all cases of severe cervical intraepithelial neoplasia, the type of HPV present in a given specimen could not be predicted on the basis of morphologic, immunocytochemical, or electron microscopic findings. It is concluded that virtually all dysplastic lesions of the cervix contain HPV DNA, that HPV is thus likely to be a major etiologic agent in the pathogenesis of cervical dysplasia, and that histopathologic features are not predictive of HPV type.

Antigens, Viral↗
PubMed1986

Hepatitis B virus DNA contains a glucocorticoid-responsive element.

It has recently been shown that hepatitis B virus (HBV) contains a transcriptional enhancer element. In order to determine whether this enhancer responds to glucocorticoids, a series of derivatives of plasmid pA10CAT2 was constructed containing the HBV enhancer and variable lengths of further upstream sequences. Transient expression of chloramphenicol acetyltransferase (CAT) was determined after introduction of these plasmids into PLC/PRF/5, Hep 3B, Hep G2, HeLa, and mouse L cells. Highest CAT activity was noted in the human hepatocellular carcinoma line PLC/PRF/5, which contains integrated HBV DNA sequences. Dexamethasone augmented CAT expression in all cell lines tested with 40% of maximal induction at 10 nM and maximum stimulation (3- to 8-fold) at 1 microM dexamethasone. Dexamethasone augmentation of CAT expression was observed only when constructs contained HBV DNA sequences residing upstream to map position 735 from the EcoRI site. This indicates that the glucocorticoid-responsive region is distinct from the previously defined HBV enhancer sequence located at map position 1080-1234. These studies suggest that HBV DNA contains a glucocorticoid-responsive element, which may mediate expression of HBV genes in infected mammalian cells.

Acetyltransferases↗
PubMed1985

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA.

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.

Animals↗
PubMed1985

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.

Adult↗