Biomedical subjects

R A Spence

Publications and source records attributed to R A Spence.

At least 19 recordsLinked to original sources

PubMed2001

Lysine(164)alpha of protein farnesyltransferase is important for both CaaX substrate binding and catalysis.

Protein farnesyltransferase (FTase) catalyses the formation of a thioether linkage between proteins containing a C-terminal CaaX motif and a 15-carbon isoprenoid. The involvement of substrates such as oncogenic Ras proteins in tumour formation has led to intense efforts in targeting this enzyme for development of therapeutics. In an ongoing programme to elucidate the mechanism of catalysis by FTase, specific residues of the enzyme identified in structural studies as potentially important in substrate binding and catalysis are being targeted for mutagenesis. In the present study, the role of the positive charge of Lys(164) of the alpha subunit of FTase in substrate binding and catalysis was investigated. Comparison of the wild-type enzyme with enzymes that have either an arginine or alanine residue substituted at this position revealed unexpected roles for this residue in both substrate binding and catalysis. Removal of the positive charge had a significant effect on the association rate constant and the binding affinity of a CaaX peptide substrate, indicating that the positive charge of Lys(164)alpha is involved in formation of the enzyme (E).farnesyl diphosphate (FPP).peptide ternary complex. Furthermore, mutation of Lys(164)alpha resulted in a substantial decrease in the observed rate constant for product formation without alteration of the chemical mechanism. These and additional studies provide compelling evidence that both the charge on Lys(164)alpha, as well as the positioning of the charge, are important for overall catalysis by FTase.

Alkyl and Aryl Transferases↗

PubMed2000

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition.

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.

Alkyl and Aryl Transferases↗

PubMed2000

Sportsman's hernia.

BACKGROUND: Sportsman's hernia is a debilitating condition which presents as chronic groin pain. A tear occurs at the external oblique which may result in an occult hernia. The definition, investigation and treatment of this condition remain unclear. METHODS: A systematic Medline search was performed and all literature pertaining to chronic groin pain, groin injury, sportsman's hernia and sportsman's groin from 1962 to 1999 was retrieved for analysis. RESULTS: The costs of computed tomography and magnetic resonance imaging are such that their routine use for assessment of patients with groin pain cannot be justified. They may, however, be employed in difficult cases to help define the anatomical extent of a groin injury. Plain radiography, ultrasonography and scintigraphy should be the usual first-line investigations to supplement clinical assessment. Herniography may help in situations of obscure chronic groin and pelvic pain. There is no consensus view supporting any particular surgical procedure for sportsman's hernia. A number of reports have been published describing different repairs of the posterior inguinal wall deficiency. Appropriate repair of the posterior wall results in therapeutic benefit in selected cases. CONCLUSION: The diagnosis of sportsman's hernia is difficult. The condition must be distinguished from the more common osteitis pubis and musculotendinous injuries. Early surgical intervention is usually, although not always, successful when conservative management has failed.

Athletic Injuries↗

PubMed1999

The role of fine needle aspiration cytology (FNAC) in the investigation of superficial lymphadenopathy; uses and limitations of the technique.

Aspirates (n = 163) from 157 patients with enlarged superficial lymph nodes were obtained over a 5-year period in a combined surgical/FNAC clinic. A definitive diagnosis was achieved in over 77% of the cases: benign 52.7%, malignant 25.1%. The diagnostic accuracy was 94.4%, sensitivity 85.4%, and specificity 100%. The false-negative rate was 12.5% but decreased to 3.5% when lymphoma cases were excluded. There were 36 cases of metastatic disease, the majority of which were from a primary breast carcinoma. The main diagnostic difficulty was in distinguishing low-grade lymphoma from reactive hyperplasia. An added advantage was that aspirated material could be used in ancillary tests to help with the differential diagnosis. FNAC has a well-defined role in the investigation of superficial lymphadenopathy. Used in the proper setting it will provide a definitive diagnosis in the majority of cases, especially relating to recurrent malignancy or metastatic disease. Patients with a reactive cytological picture and no clinically suspicious symptoms could be spared unnecessary surgery and reviewed through follow up. This technique is cost-effective, of high diagnostic accuracy, and results in considerable resource savings.

Adolescent↗

PubMed1998

A 3-year audit of thyroid fine needle aspirates.

Fine needle aspiration (FNA) cytology is an important adjunct to the preoperative management of thyroid swellings. It is a simple and highly cost effective procedure with a higher sensitivity for the detection of malignancy than ultrasound and radio-isotope scans. We audited a total of 208 aspirates performed over a 3-year period. In our experience 61% of solitary thyroid swellings can be diagnosed confidently on FNA cytology as either non-neoplastic (46%) or neoplastic (15%). Based on these two groups the sensitivity is 91% and specificity 94%. The specificity, however, decreases to 40% if the group reactive vs neoplastic, which comprises 10-15% of cases and requires surgical intervention, is included. The positive predictive value for malignant disease is 94% while the false-negative and -positive rates excluding the unsatisfactory and reactive vs neoplastic groups are low at 2.7% and 5.4%, respectively (involving non-neoplastic lesions and benign tumours only). There was no case of malignant cytology with a benign biopsy follow up.

Biopsy, Needle↗

PubMed1997

Thyroid hormones and selenium status in breast cancer.

Within a case-control study of postmenopausal breast cancer patients (n = 99) and matched healthy controls (n = 105), thyroid hormone levels were compared and correlated with toenail selenium concentrations. Plasma triiodothyronine (T3) was significantly lower in cases (1.4 +/- 0.4 nmol/l) than in controls (1.6 +/- 0.4 nmol/l), and a strong inverse relationship with breast cancer was observed with an odds ratio of 0.17 (95% CI = 0.08-0.36) in the highest compared with the lowest tertile of T3. Plasma thyroxine and thyroid-stimulating hormone concentrations were similar between cases and controls. Plasma T3 concentration was positively associated with toenail selenium in cases (age-adjusted regression coefficient = 0.049) and controls (age-adjusted regression coefficient = 0.036). Toenail selenium concentrations tended to be lower in cases than in controls, but the differences did not reach statistical significance. Although the disease process per se may explain the lower plasma T3 concentrations, it is also possible, inasmuch as these patients were in early-stage breast cancer, that selenium status may be influencing T3 levels via changes in the activity of the selenoenzyme type 1 iodothyronine deiodinase.

Aged↗

PubMed1997

DNA denaturation sensitivity may invalidate bromodeoxyuridine--DNA flow cytometric analysis of potential doubling times in colorectal tumours.

BACKGROUND: Flow cytometric analysis of 5-bromo-2'-deoxyuridine (BrdU)-substituted DNA has been used to calculate tumour cell proliferation rate. In this methodology, DNA denaturation, commonly by hydrochloric acid, is essential to expose incorporated BrdU for quantification with monoclonal antibodies. This study was designed to establish the validity of this technique by examining the change in flow cytometric DNA profiles introduced by DNA denaturation procedures. METHODS: Four experiments were performed using suspensions of nuclei derived from human colorectal tumours exposed in vivo to 150 mg/m2 BrdU 8-11 h before sampling. RESULTS: After denaturation with hydrochloric acid 2 mol/l a significant decrease was observed in the DNA aneuploid G1 population (P < 0.001) with a concurrent increase in the DNA aneuploid S phase fraction (P < 0.05). These changes were independent of the washing-centrifugation step and were maximal at different hydrochloric acid concentrations for different tumours. CONCLUSION: Hydrochloric acid denaturation introduces a tumour-specific non-linear variation in the analysis of BrdU.

Aged↗

PubMed1997

A 3 year audit of fine needle aspirates from a symptomatic breast clinic.

A total of 2431 fine needle aspirates of symptomatic breast lumps was performed on 2096 patients over the last three years at the weekly head, neck and breast clinic at the Belfast City Hospital Trust. Diagnostic accuracy was achieved within the recommended standards although the "insufficient" rate was high at 31.8%. False negative and positive rates were low and the positive predictive value for malignancy was 99%. Excision biopsy for benign breast disease had decreased by almost a third during this period. Fine needle aspiration cytology is a highly accurate and cost-effective technique for the investigation of symptomatic breast lumps and results in significant savings.

Biopsy, Needle↗

PubMed1996

HIV-1 reverse transcriptase resistance to nonnucleoside inhibitors.

The parameters governing the polymerization mechanism of reverse transcriptase containing the tyrosine to cysteine mutation at position 181 (Y181C) were determined using pre-steady-state techniques. The pathway for single nucleotide incorporation catalyzed by Y181C is similar to that determined for wild-type RT where a rate-limiting conformational change precedes fast chemistry and is followed by slow steady-state release of the primer/template. The Y181C mutant enzyme binds a 25/45-mer duplex DNA tightly with a Kd of 11 nM. However, the Y181C mutation weakens the nucleotide affinity 2-3-fold relative to the wild-type complex. We also determined the parameters governing the mechanism of nonnucleoside inhibitor resistance with Y181C. The Kd value of Nevirapine with the mutant E.DNA complex increased approximately 500-fold. The decreased affinity of Nevirapine for the mutant enzyme is a consequence of a faster inhibitor dissociation rate from the enzyme complex of Y181C relative to that of the wild-type. The E.DNA complex of Y181C may be saturated with Nevirapine, and the I.E.DNA complex is capable of a maximum incorporation rate of 0.1 s-1 (a 10-fold faster rate than that of the wild-type I.E.DNA complex). The overall two-step binding of nucleotide to Y181C in the presence of Nevirapine remains unaffected.

Antiviral Agents↗

PubMed1996

Self-expanding stents for malignant dysphagia.

Self-expanding metallic stents have been employed successfully for vascular, urethral, and biliary strictures. In a prospective study we examined the efficacy of the 16 mm Wallstent for palliation of malignant dysphagia. Eight patients with severe dysphagia due to advanced primary (four) or secondary (four) oesophageal malignant disease were recruited and nine Wallstents were inserted (one patient required two). Dysphagia was reduced in all but one patient, who died after oesophageal perforation; a second patient had a self-limiting bout of haematemesis. Two patients required subsequent treatment for tumour ingrowth but five had no further palliative therapy from stent insertion to time of death. With careful patient selection and skillful application, a 16 mm self-expanding metal endoprosthesis affords effective palliation in malignant oesophageal obstruction.

Adult↗

PubMed1996

An audit of thyroid surgery in a general surgical unit.

A total of 143 patients undergoing thyroid surgery in a general surgical unit over an 8-year period were reviewed. In only two patients did thoracic inlet views or thyroid function tests alter clinical management. Fine-needle aspiration failed to detect one well-differentiated follicular carcinoma (false-negative rate 1.1%). The sensitivity for malignancy of fine-needle aspiration, ultrasound and radioisotope scan were 94%, 53% and 24%, respectively. The corresponding specificity was 59%, 72% and 58% and accuracy 65%, 70% and 49%, respectively. The specificity of fine-needle cytology for detecting neoplastic disease (adenoma or carcinoma) was 86% and accuracy 91%. Combinations of fine-needle cytology, ultrasound and radioisotope scanning increased the sensitivity for malignancy, so that fewer tumours were missed, but at the cost of reduced specificity, positive predictive value and accuracy. Hence, ultrasound was only recommended when fine-needle aspiration was inconclusive. Overall perioperative morbidity was 6.3% (one case of postoperative bleeding, two wound infections, four cases of prolonged hypocalcaemia). There were two proven cases of transient, but no permanent, recurrent laryngeal nerve injuries as a result of surgery. Thyroid surgery may be performed satisfactorily by general surgeons with an interest in thyroid disease. Fine-needle cytology is the most informative preoperative investigation. Although aspiration cytology, ultrasound, and scintigraphy all have appropriate indications and limitations, there is no single test or group of tests that can substitute for careful clinical assessment and follow-up.

Adolescent↗

PubMed1996

Hereditary breast cancer in Northern Ireland.

The aim of this investigation was to document hereditary breast cancer in Northern Ireland. Family history details from over nine hundred women were obtained by postal survey and one hundred and twenty nine home visits were carried out to collect pedigree information. The families documented varied in the number of affected women from three, which was the minimum criteria for inclusion, to a maximum of nine and many families described other features of hereditary disease such as bilateral breast cancer, ovarian and gastrointestinal malignancies.

Breast Neoplasms↗

PubMed1995

Coronavirus protein processing and RNA synthesis is inhibited by the cysteine proteinase inhibitor E64d.

Mouse hepatitis virus strain A59 (MHV-A59) encodes within the 22-kb gene 1 a large polyprotein containing three proteinase domains with proven or predicted cysteine catalytic residues. E64d, a specific, irreversible inhibitor of cysteine (thiol) proteinases, inhibits the processing of the gene 1 polyprotein. Specifically, E64d blocks the carboxy-terminal cleavage of p65. E64d also inhibits replication of MHV-A59 in murine DBT cells in a dose-dependent manner, resulting in reduced virus titers and viral syncytia formation. This inhibition of replication is associated with a rapid shutoff of new viral RNA synthesis, in a manner similar to that seen in the presence of cycloheximide. The E64d-associated inhibition of RNA synthesis likely results from E64d-specific inhibition of processing of the gene 1 polyprotein, resulting in inactive proteinase or replicase proteins. These results indicate that processing of the MHV-A59 gene 1-encoded polyprotein is required throughout infection to sustain RNA synthesis and virus replication.

Animals↗

PubMed1995

Mechanism of inhibition of HIV-1 reverse transcriptase by nonnucleoside inhibitors.

The mechanism of inhibition of HIV-1 reverse transcriptase by three nonnucleoside inhibitors is described. Nevirapine, O-TIBO, and CI-TIBO each bind to a hydrophobic pocket in the enzyme-DNA complex close to the active site catalytic residues. Pre-steady-state kinetic analysis was used to establish the mechanism of inhibition by these noncompetitive inhibitors. Analysis of the pre-steady-state burst of DNA polymerization indicated that inhibitors blocked the chemical reaction, but did not interfere with nucleotide binding or the nucleotide-induced conformational change. Rather, in the presence of saturating concentrations of the inhibitors, the nucleoside triphosphate bound tightly (Kd, 100 nM), but nonproductively. The data suggest that an inhibitor combining the functionalities of a nonnucleoside inhibitor and a nucleotide analog could bind very tightly and specifically to reverse transcriptase and could be effective in the treatment of AIDS.

Antiviral Agents↗

PubMed1995

Neoplastic lesions of the appendix.

Neoplasms occur in 0.5 per cent of appendices. Ultrasonography or computed tomography is beneficial, but preoperative detection is rare. At operation, the diagnosis is considered in under half of cases. Mucocele, localized pseudomyxoma peritonei, benign tumours and most appendiceal carcinoids are cured by appendicectomy alone. Right hemicolectomy is indicated for: (1) invasive adenocarcinoma; (2) tumours close to the caecum; (3) lesions larger than 2 cm; (4) mucin production; (5) invasion of the lymphatics, serosa or mesoappendix; and (6) cellular pleomorphism with a high mitotic rate. Tumours of 1-2 cm, small mucinous carcinoids, adenocarcinoma confined to the mucosa, and tumours in children may be treated by appendicectomy alone at the surgeon's discretion. The 5-year survival rate associated with classical carcinoid is more than 90 per cent. The prognosis of mucinous carcinoid is intermediate between that of classical carcinoid and well differentiated adenocarcinoma. The prognosis of adenocarcinoma is determined by Dukes' stage and is similar, stage for stage, to that of colorectal carcinoma.

Adenocarcinoma↗

PubMed1995

Hereditary breast cancer and linkage analysis to BRCA1.

In this study, over 1000 women with breast cancer were contacted by questionnaire and 164 families with a strong history of breast cancer were identified. Home visits to obtain full pedigree details took place for 123 women, and members of 24 families gave venous blood samples. The extracted DNA was typed by polymerase chain reaction amplification and the derived haplotypes submitted to linkage analysis which confirmed that in 12 families breast cancer susceptibility could be traced to BRCA1. The study demonstrates the number of women at risk of breast cancer in the population, illustrates the complexity of genetic analysis for hereditary breast cancer and demonstrates the problems of predictive analysis in clinical management.

Breast Neoplasms↗

PubMed1995

An audit of surgery of the parotid gland.

The management of patients undergoing 50 surgical procedures to the parotid gland was reviewed. The overall accuracy of fine needle aspiration cytology was 87%, false-positive and false-negative rates for malignant disease both being 4%. The sensitivity, specificity and accuracy of fine needle cytology for malignant parotid tumours was 66%, 95%, and 91%, respectively, that of benign tumours (pleomorphic adenoma or Warthin's tumour) being 88%, 83% and 87%, respectively. Sensitivity, specificity and accuracy for the remaining (principally inflammatory) parotid diseases was 100%, 95% and 96%, respectively. The predictive value of a positive test for malignant tumours, benign tumours and inflammatory conditions was 66%, 94% and 75%, respectively. The negative predictive value for these conditions was 95%, 71% and 100%, respectively. Facial nerve weakness after parotidectomy occurred in three patients (8.8%), being permanent in two cases (both malignant). Although Frey's syndrome was not recorded in any of the notes, careful follow-up revealed two cases (6%). To date there have been no local recurrences after excision of either benign or primary malignant parotid masses. One patient with squamous cell carcinoma metastatic to the parotid gland died, despite block dissection of the neck and radiotherapy. This small series with a limited follow-up suggests that diseases of the parotid gland can be managed by general surgeons with an interest in this field. Although fine needle aspiration and ultrasonic scan may be helpful, the decision to operate should be made on clinical grounds.

Adolescent↗

PubMed1994

Malignant anal tumours.

Anal tumours represent 5 per cent of anorectal cancers and exist as two clinical entities: tumours of the anal canal and those of the anal margin. Smoking and sexual behaviour, particularly homosexual anal intercourse, are important aetiological factors. This association is related to anal warts and human papillomavirus infection, notably type 16, which is found in around 70 per cent of warts. Symptoms are non-specific and are frequently attributed to benign conditions. Rectal examination reveals a characteristically infiltrating lesion and any suspicious anal area should be biopsied. There are two histological types. Squamous carcinoma comprises approximately 95 per cent of anal tumours and includes the 35 per cent of tumours derived from the anal transition zone (cloacogenic tumours), containing a mixture of squamous and mucinous elements. The remaining 5 per cent of anal tumours are adenocarcinoma. Squamous cell tumours of the anal canal are probably best treated using radiotherapy (with chemotherapy) as complete response rates, 5-year survival rates, and incidences of normal sphincter function and significant toxicity are around 80, 70, 75 and 20 per cent respectively. Treatment failures may be salvaged by surgery. The 5-year survival and local recurrence rates for radical surgery are around 60 and 25 per cent respectively; there are few indications for local excision. In contrast, 60 per cent of anal margin tumours are suitable for local excision, the 5-year survival rate being in excess of 80 per cent. Combining radiotherapy with surgery may give additional benefit. Current randomized controlled trials should further clarify the relative merits and demerits of the treatment options.

Anus Neoplasms↗