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P R Blake

Publications and source records attributed to P R Blake.

At least 37 records · Page 2Linked to original sources
PubMed1992

Solution-state structure by NMR of zinc-substituted rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The three-dimensional solution-state structure is reported for the zinc-substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. Structures were generated with DSPACE by a hybrid distance geometry (DG)-based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (NOE)-derived interproton distance restraints, including 67 interresidue, 124 sequential (i-j = 1), 75 medium-range (i-j = 2-5), and 137 long-range (i-j > 5) restraints. All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 A), and upper bounds of 2.7 A, 3.3 A, and 5.0 A were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty-three backbone-backbone, six backbone-sulfur (Cys), two backbone-side chain, and two side chain-side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to > 16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridium pasteurianum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; +/- standard deviation) of 0.024 +/- 0.003 A2 and a maximum total penalty of 0.035 A2. Superposition of the backbone atoms (C, C alpha, N) of residues A1-L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (+/- SD) of 0.42 +/- 0.07 A. Superposition of all heavy atoms for residues A1-L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 +/- 0.08 A. Qualitative comparison of back-calculated and experimental two-dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X-ray crystallography for native Rd from the mesophilic organism C. pasteurianum, with the average rms deviation value for backbone atoms of residues A1-L51 of P. furiosus Zn(Rd) superposed with respect to residues K2-V52 of C. pasteurianum Rd of 0.77 +/- 0.06 A. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Comparison of the X-ray structure of native rubredoxin from Pyrococcus furiosus with the NMR structure of the zinc-substituted protein.

The three-dimensional X-ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at -161 degrees C, and the NMR structure of the zinc-substituted protein, determined in solution at 45 degrees C, are compared. The NMR and X-ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.

Amino Acid Sequence↗
PubMed1992

Quantitative measurement of small through-hydrogen-bond and 'through-space' 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin from Pyrococcus furiosus.

A method is described for measurement of small unresolvable heteronuclear J couplings. The method is based on quantitative analysis of a phase-purged heteronuclear spin-echo difference spectrum, and is demonstrated for measuring 1H-113Cd and 1H-199Hg J couplings in metal-substituted rubredoxin (M(r) approximately 5.4 kDa) from Pyrococcus furiosus. Couplings from cadmium to backbone amide protons that are hydrogen bonded to the Cys-S atoms directly bonded to Cd vary from smaller than 0.3 to 1.8 Hz; a 'through-space' coupling between Cd and the protons of an alanine methyl group was measured to be 0.3 Hz. Couplings to 199Hg are significantly larger and fall in the 0.4-4 Hz range.

Amino Acid Sequence↗
PubMed1992

The role of routine urine and parametrial aspirate cytology in staging carcinoma of the cervix.

The role of routine urine and parametrial aspirate cytology as a means of refining the staging of carcinoma of the cervix was assessed in a series of 45 consecutive patients undergoing examination under anaesthetic and cytoscopy. Only 2/45 (4%) of urine samples were positive and 3/37 (8%) of parametrial aspirates were abnormal. In none of these patients was additional information obtained over that already available from cytoscopy, digital examination and routine radiology. These procedures have no role in the routine clinical staging of cervical carcinoma.

Biopsy, Needle↗
PubMed1991

Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR.

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.

Amino Acid Sequence↗
PubMed1991

C-terminal retroviral-type zinc finger domain from the HIV-1 nucleocapsid protein is structurally similar to the N-terminal zinc finger domain.

Two-dimensional NMR spectroscopic and computational methods were employed for the structure determination of an 18-residue peptide with the amino acid sequence of the C-terminal retroviral-type (r.t.) zinc finger domain from the nucleocapsid protein (NCP) of HIV-1 [Zn(HIV1-F2)]. Unlike results obtained for the first retroviral-type zinc finger peptide, Zn(HIV1-F1), [Summers et al. (1990) Biochemistry 29, 329], broad signals indicative of conformational lability were observed in the 1H NMR spectrum of Zn-(HIV1-F2) at 25 degrees C. The NMR signals narrowed upon cooling to -2 degrees C, enabling complete 1H NMR signal assignment via standard two-dimensional (2D) NMR methods. Distance restraints obtained from qualitative analysis of 2D nuclear Overhauser effect (NOESY) data were used to generate 30 distance geometry (DG) structures with penalties (penalty = sum of the squared differences between interatomic distances defined in the restraints file and in the DG structures) in the range 0.02-0.03 A2. All structures were qualitatively consistent with the experimental NOESY spectrum based on comparisons with 2D NOESY back-calculated spectra. Superposition of the backbone atoms (C, C alpha, N) for residues C(1)-C(14) gave pairwise RMSD values in the range 0.16-0.75 A. The folding of Zn(HIV1-F2) is very similar to that observed for Zn(HIV1-F1). Small differences observed between the two finger domains are localized to residues between His(9) and Cys(14), with residues M(11)-C(14) forming a 3(10) helical corner.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Hormone replacement therapy and survival after surgery for ovarian cancer.

OBJECTIVE: To evaluate whether hormone replacement therapy affects survival in women who have undergone bilateral salphingo-oophorectomy because of epithelial ovarian cancer. DESIGN: Retrospective analysis by review of patients' notes and questionnaires completed by general practitioners to compare the overall survival and disease free survival in patients with ovarian cancer who did or did not receive hormone replacement therapy after diagnosis. Data were analysed by Cox regression, with hormone replacement therapy as a time dependent covariate because patients who received hormone replacement did so at different times after diagnosis. SETTING: Gynaecological oncology unit of Royal Marsden Hospital. PATIENTS: 373 patients aged 50 years or younger who attended the hospital from 1972 to 1988. All of the women had undergone bilateral salpingoophorectomy for epithelial ovarian cancer. In all, 78 had received hormone replacement therapy, starting at a median of four months after diagnosis. INTERVENTION: A questionnaire was sent to the general practitioners of all patients who were not recorded as having received hormone replacement therapy. MAIN OUTCOME MEASURES: Overall survival and disease free survival. RESULTS: There was no significant difference in survival between women receiving hormone replacement therapy and those not receiving it after accounting for the effects of other known prognostic factors (stage of cancer, differentiation of tumour, histological results, and time to relapse). The relative risk of dying in those who received hormone replacement therapy was 0.73 (95% confidence interval 0.44 to 1.20). In addition, there was no significant difference in disease free survival (relative risk in those receiving hormone replacement therapy was 0.90; 95% confidence interval 0.52 to 1.54). CONCLUSIONS: This study shows that hormone replacement therapy is unlikely to have a detrimental effect on the prognosis of patients with ovarian cancer, but this would be shown conclusively only by a randomised controlled trial.

Adult↗
PubMed1991

1,3,8-trisubstituted xanthines. Effects of substitution pattern upon adenosine receptor A1/A2 affinity.

A series of 11 8-substituted xanthines having three different substitution patterns on the 1- and 3-positions [pattern a (R1 = R3 = CH2CH2CH3), b (R1 = CH2CH2CH3, R3 = CH3), and c (R1 = CH3, R3 = CH2CH2CH3)] was prepared. These compounds were assessed for affinity and selectivity in binding to adenosine A1 and A2 receptors. Compounds with greatest affinity at the A1 receptor had the 1,3-substitution pattern a. With one exception, compounds with pattern a also exhibited the most potent binding at the A2 receptor; however, several compounds with pattern c were equipotent at the A2 receptor with those having pattern a. Additionally, the substituents on the 1- and 3-positions of these 8-substituted xanthines were equally important for determining maximum affinity to the A1 receptor, while the substituent at the 3-position is more important than the substituent at the 1-position for potency at the A2 receptor. As a result of this, it is possible to maximize selectivity for the A1 receptor by choice of the 1- and 3-position substituents. However, the R1/R3 substitution pattern required for maximum A1 selectivity is also dependent upon the substituent in the 8-position in a manner which is not fully understood.

Animals↗
PubMed1991

A new treatment for endometrial cancer with gonadotrophin releasing-hormone analogue.

OBJECTIVE: To test the antitumour effect of gonadotrophin releasing-hormone (GnRH) analogues in women with recurrent endometrial cancer. DESIGN: An open phase II observational trial of GnRH analogues. Serial measurements of gonadotrophins, sex hormones and tumour dimensions were made together with repeat biopsy when possible to assess the response to treatment. SETTING: The outpatient clinics of the Department of Medical Oncology at The Royal London, Royal Marsden and St Bartholomew's hospitals. SUBJECTS: 17 patients with endometrial cancer which had recurred after surgery, radiotherapy and progesterone treatment and was symptomatic, progressive and assessable for response. INTERVENTION: Monthly subcutaneous injection of GnRH analogue. MAIN OUTCOME MEASURES: Reduction in serum gonadotrophins and reduction in tumour dimensions. RESULTS: Six out of 17 patients (35%, 95% CI 12.6-58%) achieved a complete or partial remission which continues for a median of 20 months with no adverse effects. CONCLUSION: GnRH analogues have a significant antitumour effect in recurrent endometrial cancer which warrants further examination in comparison with progestogens.

Adenocarcinoma↗
PubMed1990

The nucleocapsid protein isolated from HIV-1 particles binds zinc and forms retroviral-type zinc fingers.

The role of zinc in retroviral gag protein function has been addressed through the application of high-resolution nuclear magnetic resonance spectroscopy to samples of the nucleocapsid protein (NCP, p7) isolated directly from infectious HIV-1 particles. Unlike reports for the NCP from avian myeloblastosis virus (AMV) particles [Jentoft et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7094], we find that the HIV-1 NCP binds 2 equiv of zinc tightly and stoichiometrically. Two-dimensional NMR spectroscopic studies reveal that zinc binding induces formation of folded domains that are conformationally similar to (if not identical with) structures observed previously for relevant retroviral-type (RT) zinc finger peptides [formerly called zinc fingerlike peptides; Summers et al. (1990) Biochemistry 29, 329]. This finding is consistent with the hypothesis that the inability of mutant proteins (with substituted Cys and His residues) to package viral RNA results from deficient zinc-binding capability, which may have significant consequences in the development of vaccines for the prevention of AIDS.

Amino Acid Sequence↗
PubMed1990

Carboplatin and ifosfamide in ovarian cancer phase II and III trials. London Gynaecological Oncology Group.

Over the past few years controversy has continued as to whether alkylating agents such as cyclophosphamide or chlorambucil are as effective as cisplatin in advanced ovarian cancer. Arguments have also been put forward against the use of combination chemotherapy, which is clearly more toxic than single-agent treatment and is probably no more effective than a single-agent platinum compound except in terms of producing a higher response rate. Certainly survival is not improved.

Antineoplastic Agents↗
PubMed1990

The value of cervical cytology in detecting recurrent squamous carcinoma of the cervix postradiotherapy.

Twenty-six patients with central recurrent squamous carcinoma of the cervix following radiotherapy were identified from hospital records, 22 of which had cervical smears available for review, and are described here. As a second part to this study the cervical cytology results for 1987 and 1988 were compared with the clinical findings and eventual outcome. From a total of 384 smears in 1987 and 1988 there were 11 correctly identifying recurrence. Two smears, however, were false positive and two false negative. The overall sensitivity was 85% with a specificity of 99.5%. In addition three microscopic, operable recurrences were identified by cytology. In experienced hands routine smear cytology postradiotherapy is a reliable and useful addition to surveillance.

Carcinoma, Squamous Cell↗
PubMed1989

The role of lymphography in the management of carcinoma of the endometrium.

In order to assess the role of lymphography in the postoperative management of carcinoma of the endometrium, 57 consecutive cases were analysed retrospectively. Forty-eight patients had undergone definitive surgery and bipedal lymphography was performed in 41 cases (85%). Unilateral pelvic lymphadenopathy was detected in three cases (7%); two stage I, one stage II. In no case did the result of lymphography alter management, based on histological criteria, with either intracavitary radiotherapy alone or in combination with external beam therapy to the pelvis. In a short median follow-up of 11 months (range 2-19 months), four cases have relapsed but all had normal lymphography at initial postoperative staging. Bipedal lymphography has no role in the routine management of carcinoma of the endometrium.

Adult↗
PubMed1986

Combined radiotherapy and chemotherapy for advanced carcinoma of the cervix.

Until recently the use of cytotoxic drugs in the treatment of squamous carcinoma of the cervix has been limited to patients with local recurrence after primary therapy or with distant metastases. The effectiveness of some cytotoxic drugs, especially cisplatin, in producing tumour regression in this group of patients with a poor prognosis, has encouraged the use of chemotherapy at an earlier stage of the disease process. Since 1979 patients with FIGO IIb squamous carcinoma of the cervix extending more than half-way to the pelvic side-wall and Stages III and IV disease have been treated with a combination of cisplatin or methotrexate and radiotherapy. The complete remission rate of Stage III tumours was 89% with a local recurrence rate of only 8%. In patients with advanced Stage IIb disease only 44% achieved complete remission and three patients with Stage IV disease failed to respond. The projected 5-year survival rate is 66% for patients with Stage III disease and 49% for all patients. The results of this combined treatment are presented and discussed and the suggestion made that chemotherapy combined with radiotherapy can be safely used to advantage in patients at high risk of local relapse.

Adult↗