Biomedical subjects
M E Black
Publications and source records attributed to M E Black.
A single amino acid substitution abolishes feedback inhibition of vaccinia virus thymidine kinase.
Exploitation of differences in the substrate specificity of the type I and type II thymidine kinases (EC 2.7.1.21, TK) expressed by the Herpesviridae and Poxviridae (and human cells), respectively, has lead to the development of effective antiherpetic drugs such as acyclovir and gancyclovir. Analysis of type I TK protein sequences reveals a consensus sequence which corresponds to domain IV of type II TK proteins such as that encoded by vaccinia virus (VV). The type I descriptor (Xpho - + + Xpho) differs at the second position from the type II consensus sequence (Xpho Xphi + + Xpho) by having an aspartic acid residue (D) substituted for a glutamine (Q). To test the hypothesis that this substitution may be responsible for the observed differences in substrate specificity of these enzymes and as a approach to identify the nucleoside binding site of the type II VV TK, site-directed mutagenesis was employed to alter glutamine 114 (Q114) within domain IV of VV TK to a histidine (Q114H) or an aspartic acid (Q114D). All of the mutant enzymes retained full enzymatic activity as compared to wild-type VV TK when thymidine or bromodeoxyuridine were used as substrates. However, unlike the wild-type herpes simplex (type 1) TK enzyme, neither wild-type nor domain IV VV TK mutants were able to phosphorylate acyclovir or cytidine substrates. Surprisingly, the domain IV VVTK mutants displayed a dramatic loss of feedback inhibition by dTTP. Mutations of the Q114 position also lead to a difference in ATP binding as demonstrated by an altered elution pattern of Q114H and Q114D from an ATP-agarose affinity column with dTTP. Taken together, these results suggest that domain IV of VV TK is not involved directly in substrate discrimination but instead participates in feedback inhibition by dTTP.
Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase. Evidence for a potential role in magnesium binding.
Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII. Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951). In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b). With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J. (1982) EMBO J. 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press). To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84. Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability. The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack.
Utilisation by homeless people of acute hospital services in London.
OBJECTIVES: To estimate the numbers and distribution of homeless people in London; to quantify the utilisation of acute inpatient services by homeless people in two health authorities; and to predict the total numbers of admissions in homeless people in district health authorities across London. DESIGN: Data were collected from various sources on the distribution of homeless people across London boroughs. All unplanned acute inpatient admissions during November 1990 to relevant hospitals were identified. SETTING: Bloomsbury and Paddington and North Kensington, two former inner London district health authorities. SUBJECTS: Homeless people in London residing in bed and breakfast and private sector leased accommodation, residing in hostels, and of no fixed abode. MAIN OUTCOME MEASURES: Number and cost of acute unplanned admissions in homeless people in two health authorities in November 1990; predicted number of such admissions each year in district health authorities in London. RESULTS: There were at least 60,000 homeless people in London in March 1990. The majority were housed in temporary accommodation (55,412). There were at least 3295 hostel dwellers and 651 people sleeping rough. Homeless people accounted for 105 (8%) of the 1256 acute unbooked admissions in residents of Bloomsbury and Paddington and North Kensington health authorities in November 1990. Considerable variations in the pattern of acute unplanned admissions in homeless people were observed in the two districts with respect to housing status and specialty of admission. The total number of acute unplanned admissions in homeless people across London each year was estimated at 7598, ranging from 38 in Bexley to 1515 in Parkside. CONCLUSIONS: The results have fundamental implications for resource allocation across London. Allocation must take better account of the heterogeneity, uneven distribution, and extra health needs of homeless people.
Nucleotide sequence of the Escherichia coli thymidine kinase gene provides evidence for conservation of functional domains and quaternary structure.
Using lambda bacteriophage clones from the Kohara Escherichia coli library spanning minutes 25.5 to 28.5 on the E. coli chromosome (strain W3110), two overlapping DNA fragments were identified which were able to confer thymidine kinase (TK) enzyme activity to a TK- strain of E. coli (KY895). This genetic complementation assay was used in concert with subcloning procedures to identify the minimal region (a 900 bp EcoRI-SalI fragment) which contained the E. coli thymidine kinase gene (tdk). The nucleotide sequence of the EcoRI-SalI fragment and a small portion of the adjoining downstream fragment was determined. Computer analysis of the derived sequence indicated the presence of a rightward-reading open reading frame of 615 bp which was capable of encoding a 205-amino-acid polypeptide with a predicted Mr of 23458 daltons. The in vivo transcriptional activity of this locus was confirmed by Northern blot hybridization analysis of RNA isolated from E. coli JM101 or KY895 which detected a 650-nucleotide RNA transcribed from this region. This places the tdk gene at approximately minute 27.35 on the E. coli W3110 chromosome, about 15 kb downstream from the narG locus and approximately 25 kb upstream of the trp operon. Although the predicted Mr of the E. coli TK protein was 23.5 kDa, gel-filtration analyses suggested that, like eukaryotic thymidine kinases, the active form of this enzyme is a multimeric complex.(ABSTRACT TRUNCATED AT 250 WORDS)
Ivermectin in the treatment of onchocerciasis in Britain.
Thirty-one patients with onchocerciasis were treated with a single dose of 150-200 micrograms/kg ivermectin. They were observed for three days to detect acute reactions and were reassessed clinically and parasitologically three, six and twelve months after treatment. 22 patients were expatriates. All had early, light and often localised infections and were not reexposed to infection during the course of the study. 25 patients completed follow-up. Patients who relapsed were retreated with the same dose after an interval of not less than five months. Adverse reactions were less severe than those experienced by similar patients treated with diethylcarbamazine and only one patient was given corticosteroids. 17 patients (68%) relapsed within one year and two thirds of these had done so within 6 months. A similar pattern was seen after the second dose. A single dose of ivermectin, repeated every three to six months as necessary, is likely to be the treatment of choice for patients in non-endemic areas, lightly infected with Onchocerca volvulus, until a safe macrofilaricidal drug emerges. One third of such patients may be cured with each treatment.
Identification of the ATP-binding domain of vaccinia virus thymidine kinase.
Although small in size (20 kDa), the vaccinia virus (VV) thymidine kinase protein (EC 2.7.1.21 TK) is a relatively complex enzyme which must contain domains involved in binding both substrates (ATP and thymidine) and a feedback inhibitor (dTTP), as well as sequences directing the association of individual protein monomers into a functional tetrameric enzyme. Alignment of predicted amino acid sequences of the thymidine kinase genes from a variety of sources was used to identify highly conserved regions as a first step toward locating potential regions housing essential domains. A conserved domain (domain I) near the amino terminus of VV TK protein had characteristics consistent with a nucleotide-binding site. Analysis of the nucleotide substrate specificity of VV TK indicated that ATP acts as the major phosphate donor for thymidine phosphorylation while GTP, CTP, and UTP were inefficient substrates. Site-directed mutagenesis was performed on domain I to generate 11 mutant enzymes. Comparison of the wild-type and mutant proteins with regard to enzyme activity revealed that two of the mutant enzymes, T18 and S19, exhibited enhanced enzyme activity (3.73-fold and 1.35-fold, respectively) relative to the control. The other mutations introduced led to greatly reduced levels of enzyme activity which correlated with a reduced or altered ability of the mutant enzymes to bind ATP as determined by ATP-agarose affinity chromatography. Wild-type VV TK bound to an ATP affinity column could also be eluted with dTTP. Glycerol gradient separation of wild-type TK in the presence or absence of dTTP indicated that dissociation of the tetrameric complex was not the means by which enzymatic inhibition was achieved. Taken together, these results suggest that (i) domain I (amino acids 11-22) of the VV TK corresponds to the ATP-binding site, and (ii) that dTTP is able to interfere with ATP binding, either directly or indirectly, and thereby inhibit enzymatic activity without dissociating the native enzyme.
Quaternary structure of vaccinia virus thymidine kinase.
Thymidine kinase enzymes isolated from a variety of sources are generally considered to have a native molecular weight of 80-90 kDa composed of two 40-45 kDa subunits. Although these parameters may accurately describe the atypical deoxypyrimidine kinases expressed by members of the Herpesviridae, the nucleotide sequences of thymidine kinase genes isolated from human, mouse, chicken and variety of poxviruses (vaccinia virus, monkeypox virus, variola virus, fowlpox virus and capripoxvirus) predict molecular weights on the order of 20-25 kDa for the derived primary translation products. To resolve this apparent dilemma, velocity sedimentation centrifugation, gel filtration chromatography and protein cross-linking procedures were employed to provide experimental evidence that enzymatically-active vaccinia virus thymidine kinase is a homotetrameric complex of 20 kDa monomers with a native Mr of 80 kDa.
Attitudes of diploma student nurses toward adult clients.
This descriptive study was conducted to determine the attitudes of diploma nursing students towards adult clients of different ages in various health situations. The present study has attempted to fill a gap in nursing research using a new approach: "the person perception paradigm." One hundred college students in the last year of their nursing program completed a questionnaire. Students in this study rated eight photos of male clients, ranging in ages from 20 to 90 years, in various health situations. The rating scales were set up with 12 pairs of bipolar adjectives which reflected three dimensions: competence, benevolence, and health. The second part of the questionnaire consisted of seven questions pertaining to the particular respondent. The subject data sheet required the respondents to state their age, sex, current status in the diploma program, previous education, experiences with the elderly, and preference of work upon completion of the diploma nursing program. A multivariate repeated measures analysis of variance indicated that nursing students demonstrated positive attitudes toward the elderly as compared to younger clients for competence, benevolence, and health. In particular, the student nurses rated the 60- and 80-year-old men more positively than younger clients in both success and failure conditions. The magnitude of the differences for the success and failure ratings was much greater for the 20- and 60-year-old men than the magnitude of the ratings for the 40-year-old men or 80-year-old men. The success and failure interaction did not clearly support the expectation of a subtle age bias with the 60- and 80-year-old men.(ABSTRACT TRUNCATED AT 250 WORDS)
Expression vector pT7:TKII for the synthesis of authentic biologically active RNA encoding vaccinia virus thymidine kinase.
A transcription vector, pT7: TKII, was constructed by a novel application of the polymerase chain reaction. Chimeric oligodeoxynucleotides were used to direct the synthesis of a DNA fragment which consisted of a truncated bacteriophage T7 promoter element fused to the vaccinia virus (VV) thymidine kinase gene (tk). This fragment was cloned into a pUC118 plasmid and sequenced to ensure no mutations had occurred during its synthesis. When linearized at the 3' end of the VV tk gene at the BamHI site located in the polylinker region of the vector, pT7:TKII was efficiently transcribed by T7 RNA polymerase into a 595 nucleotide transcript whose 5' end was identical to that found on authentic nascent VV tk mRNA. When translated in a rabbit reticulocyte lysate system, the synthetic VV tk RNA was shown to be biologically active in that it directed the synthesis of a 20-kDa protein which assembled into an enzymatically active 80-kDa tetrameric complex which was indistinguishable from VV thymidine kinase (TK) enzyme isolated from VV-infected cells. The pT7:TKII vector provides a powerful approach with which: (i) to investigate the translational and posttranslational regulation of the VV tk gene; (ii) to use directed genetics to identify potential cis-acting regulatory sequences or structures present within the VV tk RNA; and (iii) to apply protein engineering procedures to identify the catalytic, allosteric and subunit interactive domains of the VV TK enzyme. As an example, the translational effects of adding a m7G cap structure to the pT7:TKII-derived VV tk RNA are presented.
Self help groups and professionals--what is the relationship?
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Quantum chemistry in drug research.
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The origins and status of speech therapy in the schools.
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Evaluation of the reorganization of a public health nursing division.
From 1986 to 1988, the Nursing Division of the Hamilton-Wentworth Department of Public Health Services undertook an analysis of our present organizational structure, incorporating knowledge from current literature, community trends and staff perceptions. From this analysis, a division-wide reorganization was implemented from the current practice in which the Public Health Nurse (PHN) delivered nursing care across all ages and stages (generalist role) to one organized by target populations. These events were described in an earlier article. As part of the plan for reorganization, the nurse management team implemented an evaluation process which included five components: 1) an internal staff survey; 2) an external agency survey; 3) a program-specific activity analysis; 4) a program-specific measure of knowledge change and; 5) a longitudinal job satisfaction survey. This article describes this evaluation and presents implications for the future.
A simple sequencing gel-saving tip.
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