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Biomedical subjects

A Herbert

Publications and source records attributed to A Herbert.

At least 19 recordsLinked to original sources
PubMed2001

The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1.

RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5' to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11-15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited.

Adenosine Deaminase↗
PubMed2001

Adenocarcinoma of the uterine cervix compared with squamous cell carcinoma: a 12-year study in Southampton and South-west Hampshire.

In a 12-year study of the population of Southampton and South-west Hampshire (SSWH), there was no rise or fall in the incidence of adenocarcinoma of the uterine cervix, although the incidence of squamous cell carcinoma fell from 14 to 7.2 per 100000 women years and the overall fall in age-adjusted incidence of cervical carcinoma was commensurate with that of England and Wales. The majority (59%) of adenocarcinomas were seen in women aged less than 50, supporting the concept of a higher risk in young women. Screen-detected carcinomas accounted for 50% of adenocarcinomas and 41% of squamous cell carcinomas in women aged 20-64 (the difference was not significant). There were more screen-detected adenocarcinomas of less than 3 mm depth of invasion and 7 mm lateral extension during the third period of the study (1991-1993). The results are consistent with reports of an increased risk of cervical cancer in women born since 1940, and lesser effectiveness of screening in preventing adenocarcinoma compared with squamous cell carcinoma. High prevalence of early screen-detected carcinomas may have been a factor in recent reports of increased incidence of adenocarcinoma.

Adenocarcinoma↗
PubMed2001

Personal view. Is it reality or an illusion that liquid-based cytology is better than conventional cervical smears?

Liquid-based cytology (LBC) has been heralded as the way forward for cervical screening, and as the answer to many of its problems. It is already used as a sole method of cell preparation in many private clinics in the UK. It is being used for colposcopy smears in many NHS clinics and is now being piloted for primary screening in three screening centres in England, as well as one in Scotland and one in Wales. LBC has been welcomed as a new technology because it deals with the problem of specimen adequacy at source, removing responsibility for slide preparation and fixation from the clinician or nurse. It provides uniformly well-fixed preparations that are free of inflammatory exudate and blood, and seem easier to screen than conventional smears. There are many articles in the world literature suggesting that LBC is more accurate than conventional screening, and it is thought likely to reduce the number of false negative tests. The main reasons for piloting LBC in the NHS Cervical Screening Programme (NHSCSP) lie in its potential for reducing screening times and for reducing the numbers of repeats for inadequate tests. LBC is expensive in terms of equipment, capital costs, maintenance, consumables, training, technical preparation time, transportation and disposal of liquid media. Its costs could be justified if they were offset by the money saved from reduced screening time and repeat tests, but only if its accuracy in terms of sensitivity and specificity were proven to be equal to or better than conventional cytology. Although that is generally held to be true by the public and medical profession alike, there is very little hard evidence to support it.

Female↗
PubMed2001

Detection of cervical infections in colposcopy clinic patients.

The purpose of this study was to determine if Neisseria gonorrhoeae; Chlamydia trachomatis; herpes simplex virus; cytomegalovirus; Epstein-Barr virus; human herpesviruses 6, 7, and 8; or adeno-associated virus influenced the production of cervical intraepithelial neoplasia. Two hundred thirty-one cervical smear samples were tested for the presence of the organisms by PCR. In addition, human papillomavirus types in the samples were determined by PCR and classified into cancer risk types of high, moderate, and low. There was no link with cervical intraepithelial neoplasia status and detection of herpes simplex virus, cytomegalovirus, Epstein-Barr virus, human herpesviruses 6 and 8, gonorrhea, or chlamydia. However, high-grade cervical intraepithelial neoplasia was found more frequently with mixed infection by moderate-risk human papillomavirus types and human herpesvirus 7 than with these papillomavirus types alone. The presence of human herpesvirus 7 may increase the oncogenic potential of moderate-risk human papillomavirus types.

Adolescent↗
PubMed2001

HPV detection and measurement of HPV-16, telomerase, and survivin transcripts in colposcopy clinic patients.

AIMS: To determine whether the detection of high risk human papillomavirus (HPV) types is more predictive for high grade CIN than the current cervical smear test, and whether the production and measurement of HPV type 16 (HPV-16) and cellular survivin and telomerase transcripts can be used to discriminate between cervical HPV infections that self cure and those that induce high grade lesions. METHODS: Three hundred and fifty four cervical smear samples from women attending the colposcopy clinic were tested by the polymerase chain reaction (PCR) for the presence of HPV. Transcripts for HPV-16 E6, E6*I, E6*II, E7, and L1 as well as cellular survivin, telomerase RNA component, and telomerase reverse transcriptase were measured using fluorogenic probe (Taqman) assays. RESULTS: Referral smear grades of severe or moderate showed greater positive predictive values for CIN 2/3 than did the detection of high or moderate risk HPV types. HPV-16 transcripts from E6, E6*I, E6*II, and E7 showed high predictive values for CIN 2/3, but low sensitivity. The telomerase RNA component was detected in 53 of 57 samples and telomerase reverse transcriptase was only detected in one sample, whereas survivin transcripts were detected in 40% of samples. CONCLUSIONS: The detection of HPV-16 or cellular survivin or telomerase transcripts did not accurately predict the grade of CIN in the samples. The detection of HPV risk types correlated well with the grade of CIN; however, the referral grade smear was the most accurate predictor of the severity of the lesion. Of the 35 different HPV types detected, 18 are not included in the HPV hybrid capture II commercial test kit. The use of such kits would have missed HPV infection in 4.3% of clinic patients with CIN 2/3 lesions and 15.4% with CIN 0/1.

Adult↗
PubMed2000

The zab domain of the human RNA editing enzyme ADAR1 recognizes Z-DNA when surrounded by B-DNA.

The Zab domain of the editing enzyme ADAR1 binds tightly and specifically to Z-DNA stabilized by bromination or supercoiling. A stoichiometric amount of protein has been shown to convert a substrate of suitable sequence to the Z form, as demonstrated by a characteristic change in the CD spectrum of the DNA. Now we show that Zab can bind not only to isolated Z-forming d(CG)(n) sequences but also to d(CG)(n) embedded in B-DNA. The binding of Zab to such sequences results in a complex including Z-DNA, B-DNA, and two B-Z junctions. In this complex, the d(CG)(n) sequence, but not the flanking region, is in the Z conformation. The presence of Z-DNA was detected by cleavage with a Z-DNA specific nuclease, by undermethylation using Z-DNA sensitive SssI methylase, and by circular dichroism. It is possible that Zab binds to B-DNA with low affinity and flips any favorable sequence into Z-DNA, resulting in a high affinity complex. Alternatively, Zab may capture Z-DNA that exists transiently in solution. The binding of Zab to potential as well as established Z-DNA segments suggests that the range of biological substrates might be wider than previously thought.

Adenosine Deaminase↗
PubMed2000

Cervical screening in England and Wales: its effect has been underestimated.

Opinions about cervical screening in the UK tend to follow one of two negative lines of thought. The first is that cervical cancer is a rare disease, and too much time and effort are spent on screening. The second is that it has been relatively ineffective, since incidence of invasive carcinoma did not fall until the NHS Cervical Screening Programme (NHSCSP) was introduced in 1988, although it fell by 40% since then. This paper presents publicly available data to demonstrate that neither of these views is true. Registrations of invasive carcinoma of the uterine cervix and carcinoma in situ in England and Wales between 1971 and 1996 show that a substantially increased risk of disease in women born since 1940 has been reversed, almost certainly by greatly improved screening. Cervical carcinoma is now a rare disease because most cases are prevented before they become invasive, mostly by screening young women, aged 20-40, before the decade of life when symptomatic cervical carcinoma most frequently presents.

Adolescent↗
PubMed1999

The solution structure of the Zalpha domain of the human RNA editing enzyme ADAR1 reveals a prepositioned binding surface for Z-DNA.

Double-stranded RNA deaminase I (ADAR1) contains the Z-DNA binding domain Zalpha. Here we report the solution structure of free Zalpha and map the interaction surface with Z-DNA, confirming roles previously assigned to residues by mutagenesis. Comparison with the crystal structure of the (Zalpha)(2)/Z-DNA complex shows that most Z-DNA contacting residues in free Zalpha are prepositioned to bind Z-DNA, thus minimizing the entropic cost of binding. Comparison with homologous (alpha+beta)helix-turn-helix/B-DNA complexes suggests that binding of Zalpha to B-DNA is disfavored by steric hindrance, but does not eliminate the possibility that related domains may bind to both B- and Z-DNA.

Adenosine Deaminase↗
PubMed1999

A 6 bp Z-DNA hairpin binds two Z alpha domains from the human RNA editing enzyme ADAR1.

The Z alpha domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Z alpha domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Z alpha and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (> or = 6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

Adenosine Deaminase↗
PubMed1999

Crystal structure of the Zalpha domain of the human editing enzyme ADAR1 bound to left-handed Z-DNA.

The editing enzyme double-stranded RNA adenosine deaminase includes a DNA binding domain, Zalpha, which is specific for left-handed Z-DNA. The 2.1 angstrom crystal structure of Zalpha complexed to DNA reveals that the substrate is in the left-handed Z conformation. The contacts between Zalpha and Z-DNA are made primarily with the "zigzag" sugar-phosphate backbone, which provides a basis for the specificity for the Z conformation. A single base contact is observed to guanine in the syn conformation, characteristic of Z-DNA. Intriguingly, the helix-turn-helix motif, frequently used to recognize B-DNA, is used by Zalpha to contact Z-DNA.

Adenosine Deaminase↗
PubMed1999

RNA processing in evolution. The logic of soft-wired genomes.

Direct read-out of information from DNA into RNA allows the genome to be faithfully reproduced in RNA. This outcome occurs in what may be called "hard-wired" organisms. On the other hand, in what we refer to as "soft-wired" organisms, RNA is processed extensively, allowing a number of different messages to be produced from the same gene. As a consequence, the nucleotide sequences present in RNA (referred to here as the ribotype) differ from those present in DNA (the genotype). In soft-wired organisms, RNA processing can be thought of as a series of steps, one or more of which have two mutually exclusive outcomes: a "default" outcome and an "alternative" outcome. In the presence of appropriate regulatory signals, the RNA is processed using the alternative pathway, while the default pathway is used in their absence. The setup is functionally equivalent to that found in binary "logic gates." In both cases, "logical operations" are implemented by using regulatory signals to establish a conditional relationship between input and output and can be described using the Boolean operators AND, OR, and NOT. In the case of RNA processing events, the outcomes can be used either to directly regulate cellular responses or to control other RNA processing events. In the latter case, "networks" are established that make processing of one RNA contingent on another. Such networks allow cells to respond to their surroundings by changing the connectivity between different RNA processing events, using RNA as a substrate to compute an appropriate response. As such logical operations impact phenotype, they are subject to natural selection. Through reverse transcription, successful outcomes can be incorporated into the genome.

Animals↗
PubMed1999

Proteolytic dissection of Zab, the Z-DNA-binding domain of human ADAR1.

Zalpha is a peptide motif that binds to Z-DNA with high affinity. This motif binds to alternating dC-dG sequences stabilized in the Z-conformation by means of bromination or supercoiling, but not to B-DNA. Zalpha is part of the N-terminal region of double-stranded RNA adenosine deaminase (ADAR1), a candidate enzyme for nuclear pre-mRNA editing in mammals. Zalpha is conserved in ADAR1 from many species; in each case, there is a second similar motif, Zbeta, separated from Zalpha by a more divergent linker. To investigate the structure-function relationship of Zalpha, its domain structure was studied by limited proteolysis. Proteolytic profiles indicated that Zalpha is part of a domain, Zab, of 229 amino acids (residues 133-361 in human ADAR1). This domain contains both Zalpha and Zbeta as well as a tandem repeat of a 49-amino acid linker module. Prolonged proteolysis revealed a minimal core domain of 77 amino acids (positions 133-209), containing only Zalpha, which is sufficient to bind left-handed Z-DNA; however, the substrate binding is strikingly different from that of Zab. The second motif, Zbeta, retains its structural integrity only in the context of Zab and does not bind Z-DNA as a separate entity. These results suggest that Zalpha and Zbeta act as a single bipartite domain. In the presence of substrate DNA, Zab becomes more resistant to proteases, suggesting that it adopts a more rigid structure when bound to its substrate, possibly with conformational changes in parts of the protein.

Adenosine Deaminase↗