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Biomedical subjects

R J Sanderson

Publications and source records attributed to R J Sanderson.

At least 19 recordsLinked to original sources
PubMed2000

Increased spontaneous mutation frequency in human cells expressing the phage PBS2-encoded inhibitor of uracil-DNA glycosylase.

The Ugi protein inhibitor of uracil-DNA glycosylase encoded by bacteriophage PBS2 inactivates human uracil-DNA glycosylases (UDG) by forming a tight enzyme:inhibitor complex. To create human cells that are impaired for UDG activity, the human glioma U251 cell line was engineered to produce active Ugi protein. In vitro assays of crude cell extracts from several Ugi-expressing clonal lines showed UDG inactivation under standard assay conditions as compared to control cells, and four of these UDG defective cell lines were characterized for their ability to conduct in vivo uracil-DNA repair. Whereas transfected plasmid DNA containing either a U:G mispair or U:A base pairs was efficiently repaired in the control lines, uracil-DNA repair was not evident in the lines producing Ugi. Experiments using a shuttle vector to detect mutations in a target gene showed that Ugi-expressing cells exhibited a 3-fold higher overall spontaneous mutation frequency compared to control cells, due to increased C:G to T:A base pair substitutions. The growth rate and cell cycle distribution of Ugi-expressing cells did not differ appreciably from their parental cell counterpart. Further in vitro examination revealed that a thymine DNA glycosylase (TDG) previously shown to mediate Ugi-insensitive excision of uracil bases from DNA was not detected in the parental U251 cells. However, a Ugi-insensitive UDG activity of unknown origin that recognizes U:G mispairs and to a lesser extent U:A base pairs in duplex DNA, but which was inactive toward uracil residues in single-stranded DNA, was detected under assay conditions previously shown to be efficient for detecting TDG.

Bacillus Phages↗
PubMed1999

Delayed DNA joining at 3' mismatches by human DNA ligases.

Repair synthesis catalysed by DNA polymerase beta at 1 nt gaps occurs in the main pathway of mammalian base excision repair. DNA polymerase beta has no exonucleolytic proof-reading ability, and exhibits high error frequency during DNA synthesis. Consequently, continuous correction of endogenous DNA damage by short-patch repair synthesis might lead to a high spontaneous mutation rate, unless subsequent steps in the repair pathway allow for selective removal of incorporation errors. We show here that both human DNA ligase I and III discriminate strongly between a correctly paired versus a mispaired residue at the 3' position of a nick in DNA, when assayed in the presence of physiological concentrations of KCl. The resulting delay in joining after misincorporation by DNA polymerase beta during gap filling could allow for removal of the mismatched terminal residue by a distinct 3' exonuclease.

Base Sequence↗
PubMed1999

Management of inverted papilloma via Denker's approach.

Denker's approach was used in the management of 78 patients presenting with an inverted papilloma of the nasal sinus complex between the years 1986 and 1993 at the University Teaching Hospital in Rotterdam. The recurrence rate was 9% with a mean follow-up after surgery of 56 months. There was minimal morbidity and no mortality associated with the procedure. Three patients had a squamous cell carcinoma associated with the inverted papilloma. The results of our study indicate that Denker's approach has a similar or lower recurrence rate than an open external approach to papilloma and is a safe procedure with minimal morbidity.

Carcinoma, Squamous Cell↗
PubMed1999

Increased expression of CD40 on thymocytes and peripheral T cells in autoimmunity: a mechanism for acquiring changes in the peripheral T cell receptor repertoire.

CD40, a cell surface molecule found on B lymphocytes and other antigen presenting cells, can, when engaged by CD40 ligand (CD40L), induce gene rearrangements and isotype switching. We report here that CD40 is also expressed on thymocytes and on up to 50% of peripheral T cells from autoimmune prone strains of mice. In normal animals, CD40 is present on a small population of T cells and thymocytes. CD40 is expressed on most T cell hybridomas. We demonstrate that CD40 engagement on peripheral T cells, T cell hybridomas and thymocytes results in altered TCRValpha expression. That induced expression of different Valpha's results from the activity of the recombinase gene is implied by the observation that CD40 does not induce TCR changes in RAG knock-out mice. Total cell numbers remained unchanged between anti-CD40 treated and untreated populations of thymocytes or T cells indicating that treatment does not induce cell proliferation or cell death. The data presented here suggest a mechanism by which self reactive T cells accumulate peripherally and independently of selective processes of the thymus.

Animals↗
PubMed1998

Fidelity and mutational specificity of uracil-initiated base excision DNA repair synthesis in human glioblastoma cell extracts.

The fidelity of DNA synthesis associated with uracil-initiated base excision repair was measured in human whole cell extracts. An M13mp2 lacZalpha DNA-based reversion assay was developed to assess the error frequency of DNA repair synthesis at a site-specific uracil residue. All three possible base substitution errors were detected at the uracil target causing reversion of opal codon 14 in the Escherichia coli lacZalpha gene. Using human glioblastoma U251 whole cell extracts, approximately 50% of the heteroduplex uracil-containing DNA substrate was completely repaired, as determined by the insensitivity of form I DNA reaction products to cleavage by a combined treatment of E. coli uracil-DNA glycosylase and endonuclease IV. The majority of repair occurred by the uracil-initiated base excision repair pathway, since the addition of the bacteriophage PBS2 uracil-DNA glycosylase inhibitor protein to extracts significantly blocked this process. In addition, the formation of repaired form I DNA molecules occurred concurrently with limited DNA synthesis, which was largely restricted to the HinfI DNA fragment initially containing the uracil residue and specific to the uracil-containing DNA strand. Based on the reversion frequency of repaired M13mp2 DNA, the fidelity of DNA repair synthesis at the target was determined to be about one misincorporated nucleotide per 1900 repaired uracil residues. The major class of base substitutions propagated transversion mutations, which were distributed almost equally between T to G and T to A changes in the template. A similar mutation frequency was also observed using whole cell extracts from human colon adenocarcinoma LoVo cells, suggesting that mismatch repair did not interfere with the fidelity measurements.

Bacillus Phages↗
PubMed1997

The effects of alcohol and smoking upon the age, anatomic sites and stage in the development of cancer of the oral cavity and oropharynx in females in the south west Netherlands.

A retrospective review of 303 women, aged 40 or over, with squamous cell carcinomas of the oral cavity or oropharynx was conducted in the south-west Netherlands to study the effects of smoking and alcohol upon the age of onset, site and stage of disease. It was noted that patients presenting with oropharyngeal cancers were younger and had a higher incidence of smoking and history of heavy drinking. Age at presentation was also affected by the amount of alcohol and tobacco consumed with non-users presenting with tumors approximately 15 years later. A specific finding was that heavy drinkers and smokers tended to present with late-stage-disease.

Adult↗
PubMed1997

The influence of alcohol and smoking on the incidence of oral and oropharyngeal cancer in women.

A retrospective case reference study examining the use of alcohol and tobacco in 303 women aged 40 or over suffering from oral or oropharyngeal cancer was conducted in the south-west Netherlands. Both alcohol and tobacco consumption are important in the development of oral and oropharyngeal cancer with increased consumption of both markedly increasing the risks of cancer, but alcohol having the greater effect.

Adult↗
PubMed1996

Identification of specific carboxyl groups on uracil-DNA glycosylase inhibitor protein that are required for activity.

The bacteriophage PBS2 uracil-DNA glycosylase inhibitor (Ugi) protein inactivates uracil-DNA glycosylase (Ung) by forming an exceptionally stable protein-protein complex in which Ugi mimics electronegative and structural features of duplex DNA (Beger, R. D., Balasubramanian, S., Bennett, S. E., Mosbaugh, D. W., and Bolton, P. H. (1995) J. Biol. Chem. 270, 16840-16847; Mol, C. D., Arvai, A. S., Sanderson, R. J., Slupphaug, G., Kavli, B., Krokan, H. E., Mosbaugh, D. W., and Tainer, J. A. (1995) Cell 82, 701-708). The role of specific carboxylic amino acid residues in forming the Ung.Ugi complex was investigated using selective chemical modification techniques. Ugi treated with carbodiimide and glycine ethyl ester produced five discrete protein species (forms I-V) that were purified and characterized. Analysis by mass spectrometry revealed that Ugi form I escaped protein modification, and forms II-V showed increasing incremental amounts of acyl-glycine ethyl ester adduction. Ugi forms II-V retained their ability to form a Ung.Ugi complex but exhibited a reduced ability to inactivate Escherichia coli Ung, directly reflecting the extent of modification. Competition experiments using modified forms II-V with unmodified Ugi as a competitor protein revealed that unmodified Ugi preferentially formed complex. Furthermore, unmodified Ugi and poly(U) were capable of displacing forms II-V from a preformed Ung.Ugi complex but were unable to displace Ugi form I. The primary sites of acyl-glycine ethyl ester adduction were located in the alpha2-helix of Ugi at Glu-28 and Glu-31. We infer that these two negatively charged amino acids play an important role in mediating a conformational change in Ugi that precipitates the essentially irreversible Ung/Ugi interaction.

Amino Acid Sequence↗
PubMed1995

Crystal structure of human uracil-DNA glycosylase in complex with a protein inhibitor: protein mimicry of DNA.

Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.

Binding Sites↗
PubMed1995

Processivity of Escherichia coli and rat liver mitochondrial uracil-DNA glycosylase is affected by NaCl concentration.

Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.

Animals↗
PubMed1993

Altered dental sensation following intranasal surgery.

Some patients complain of altered sensation in the mouth following nasal surgery. A prospective study of 60 consecutive patients who underwent a total of 83 intranasal procedures revealed that this was a common complication, particularly following submucous resection (SMR) or intranasal antrostomies. The area affected by pain/parasthesia appears to be anatomically related to the surgical procedure(s) performed.

Adolescent↗
PubMed1993

The assessment of alaryngeal speech.

The objective measurement of alaryngeal speech is desirable so that results between different centres can be compared. We examined several phonatory parameters of alaryngeal speech with the Visi-pitch and correlated these with the subjective assessment of voice quality. Amplitude, percentage voiced and perturbation correlated extremely well with the subjective assessment of voice quality.

Adult↗
PubMed1993

Blom-Singer prostheses--an 11 year experience of primary and secondary procedures.

Over the past 11 years, 25 primary and 29 secondary tracheo-oesophageal punctures have been performed in 51 laryngectomees. Pre-operative assessment is required for the secondary procedure. Both primary and secondary procedures require intensive speech therapy after fitting of the prosthesis. Fifty-four per cent of the primary and 37% of the secondary patients attained long-term speech, the latter figure rising to 58% with newer valves and increasing experience. Failures were commonly due to psychological or prosthesis-related problems. Complications were relatively minor, none being life threatening. Seventy per cent of the long-term users were self caring. Adequate rehabilitation counselling and support is necessary to ensure long-term use of the prosthesis.

Adult↗
PubMed1993

Otolaryngological indicator operations: one year's experience.

One year's experience of otolaryngological indicator operations is presented. Of 59 myringoplasties, 47 (79%) were successful; trainees performed 44 (74%) of the procedures. Seventy patients undergoing septal surgery reported significant improvements in nasal obstruction (P < 0.001), catarrh and facial pain (P < 0.005). Seventeen laryngectomies were performed for stage III and IV disease. The median operative time of 3.1 h rose to 5.1 h with a concomitant neck dissection. Eleven complications ensued, and the median stay for an uncomplicated laryngectomy was 16 days and 30 days following complications. Forty-one parotidectomies, 19 submandibular gland excisions and 10 ductal procedures were performed with 5 wound complications and 5 minor temporary facial nerve weaknesses. We have established a baseline for comparison of our practice with others and concluded that septal surgery gives good symptomatic relief, trainees perform reasonable numbers of myringoplasties, and improvements remain to be made in our outpatient septal surgery data collection.

Adolescent↗
PubMed1992

Tuberculoma of the nasopharynx.

A 67-year-old female patient with tuberculosis of the nasopharynx is reported. The diagnosis was confirmed on histological and bacteriological examination of a biopsy from her postnasal space. There was no evidence of any other active foci of tuberculosis but she had had a right nephrectomy 45 years previously for renal tuberculosis. A review of the literature on nasopharyngeal tuberculosis shows this to be a very rare disease in the absence of active pulmonary involvement.

Aged↗