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Biomedical subjects

H M Baker

Publications and source records attributed to H M Baker.

54 records · Page 3Linked to original sources
PubMed1994

Effects of participative decision making in central supply department: a case study.

This is an account of an effort to develop a culture of quality in a small service department of a major provincial hospital--in the belief that this could only be brought about by encouraging workers to take a larger degree of control over their work and the way in which it is carried out. The charge nurse, finding herself in a situation which amounted to a crisis in the work of the department and the lives of the staff, introduced a process which sought to establish that control. This account is derived from the journal of the charge nurse over the period of five months during which the changes occurred.

Australia↗
PubMed1993

Crystallization of the C-terminal domain of rabbit serum hemopexin.

The C-terminal domain of rabbit serum hemopexin, comprising residues 215 to 435, has been crystallized following removal of the attached carbohydrate using the endoglycosidase Endo F. The crystals, grown by vapour diffusion from solutions containing polyethylene glycol 1500, are orthorhombic, with cell dimensions a = 41.0 A, b = 64.2 A, c = 85.2 A, space group P2(1)2(1)2(1), and one molecule in the asymmetric unit. The crystals diffract to 2.4 A resolution and are suitable for X-ray structure analysis.

Animals↗
PubMed1993

Do glutathione and related enzymes play a role in drug resistance in small cell lung cancer cell lines?

Small cell lung cancer (SCLC) is treated primarily with combination chemotherapy. Despite high initial response rates, most patients eventually die with drug resistant disease. In some tumours, resistance to multiple chemotherapeutic agents is attributed to overexpression of P-glycoprotein (P-gp). However, this does not appear to be a frequent occurrence in drug resistant SCLC. Increased levels of glutathione (GSH) and related enzymes may play a role in resistance to alkylating agents as well as natural product drugs. We measured levels of GSH, glutathione S-transferase (GST), glutathione reductase (GSH Red), glutathione peroxidase (GSH Px), and gamma-glutamyl transpeptidase (gamma-GT) in a panel of 20 SCLC cell lines. Most of these lines were established from patients treated at this centre. Each cell line had a characteristic and reproducible profile of GSH and related enzyme levels. Immunoblot analysis indicated that the predominant GST in the cell lines was the anionic pi isoenzyme. The relative sensitivity of each of these cell lines to 16 different chemotherapeutic agents was measured using a modified MTT assay. Spearman rank correlation analysis was used to determine the relationships between the relative chemosensitivity of these cell lines and the levels of GSH and related enzymes. The number of positive correlations was no greater than expected by chance alone. Furthermore, there was no correlation with the treatment history of the patients from whom the cell lines were derived. These data suggest that alterations in glutathione metabolism do not play a major role in resistance to chemotherapeutic agents in these human SCLC cell lines.

Antineoplastic Agents↗
PubMed1992

Anion binding by human lactoferrin: results from crystallographic and physicochemical studies.

The anion-binding properties of lactoferrin (Lf), with Fe3+ or Cu2+ as the associated metal ion, have been investigated by physicochemical and crystallographic techniques. These highlight differences between the two sites and in the anion-binding behavior when different metals are bound. Carbonate, oxalate, and hybrid carbonate-oxalate complexes have been prepared and their characteristic electronic and EPR spectra recorded. Oxalate can displace carbonate from either one or both anion sites of Cu2(CO3)2Lf, depending on the oxalate concentration, but no such displacement occurs for Fe2(CO3)2Lf. Addition of oxalate and the appropriate metal ion to apoLf under carbonate-free conditions gives dioxalate complexes with both Fe3+ and Cu2+, except when traces of EDTA remain associated with the protein, when hybrid complexes M2(CO3)(C2O4)Lf can result. The anion sites in the crystal structures of Fe2(CO3)2Lf, Cu2-(CO3)2Lf, and Cu2(CO3)(C2O4)Lf, refined at 2.2, 2.1, and 2.2 A, respectively, have been compared. In every case, the anion is hydrogen bonded to the N-terminus of helix 5, an associated arginine side chain, and a nearby threonine side chain. The carbonate ion binds in bidentate fashion to the metal, except in the N-lobe site of dicupric lactoferrin, where it is monodentate; the difference arises from slight movement of the metal ion. The hybrid complex shows that the oxalate ion binds preferentially in the C-lobe site, in 1,2-bidentate mode, but with the displacement of several nearby side chains. These observations lead to a generalized model for synergistic anion binding by transferrins.

Binding, Competitive↗
PubMed1992

Metal substitution in transferrins: the crystal structure of human copper-lactoferrin at 2.1-A resolution.

The structural consequences of binding a metal other than iron to a transferrin have been examined by crystallographic analysis of human copper-lactoferrin, Cu2Lf. X-ray diffraction data were collected from crystals of Cu2Lf, using a diffractometer, to 2.6-A resolution, and oscillation photography on a synchrotron source, to 2.1-A resolution. The structure was refined crystallographically, by restrained least-squares methods, starting with a model based on the isomorphous diferric structure from which the ligands, metal ions, anions, and solvent molecules had been deleted. The final model, comprising 5321 protein atoms (691 residues), 2 Cu2+ ions, 2 (bi)carbonate ions, and 308 solvent molecules has good stereochemistry (rms deviation of bond lengths from standard values of 0.018 A) and gives a crystallographic R value of 0.196 for 43,525 reflections in the range 7.5-2.1-A resolution. The copper coordination is different in the two binding sites. In the N-terminal site, the geometry is square pyramidal, with equatorial bonds to Asp 60, Tyr 192, His 253, and a monodentate anion and a longer apical bond to Tyr 92. In the C-terminal site, the geometry is distorted octahedral, with bonds to Asp 395, Tyr 435, Tyr 528, and His 597 and an asymmetrically bidentate anion. The protein structure is the same as for the diferric protein, Fe2Lf, demonstrating that the closure of the protein domains over the metal is the same in each case irrespective of whether Fe3+ or Cu2+ is bound and that copper could be transported and delivered to cells equally well as iron. The differences in metal coordination are achieved by small movements of the metal ion and anion within each binding site, which do not affect the protein structure.

Binding Sites↗
PubMed1992

Establishment and characterization of a panel of human lung cancer cell lines.

The establishment and characterization of 11 human lung cancer cell lines are described in this article. Nine of these cell lines were established over a 5-year period, from 1983 to 1988, from patients treated at the Kingston Regional Cancer Centre. These include eight definite or probable small cell lung cancer (SCLC) lines and one adenocarcinoma line. In addition, two other SCLC cell lines were characterized. All of the lines have been in continuous culture for more than 2 years. The clinical histories of the patients from whom the cell lines were derived are outlined here. Several features of the cell lines are presented, including the following: (1) a comparison of the histologic features of the cell lines with the original biopsy specimens; (2) the expression of various markers, including cytokeratin, carcinoembryonic antigen, calcitonin, and neuron-specific enolase; (3) activities of the enzymes l-dopa decarboxylase and the brain isoenzyme of creatine kinase; (4) growth characteristics; (5) cloning efficiency in soft agar; (6) tumorigenicity in nude mice; and (7) cytogenetic studies. These cell lines, obtained directly from patients with a spectrum of drug-sensitive and drug-resistant tumors, will be valuable in vitro models of sensitivity and resistance to chemotherapy in lung cancer.

Adenocarcinoma↗
PubMed1992

Human melanotransferrin (p97) has only one functional iron-binding site.

The iron-binding properties of melanotransferrin, the tumour-associated antigen also known as p97, have been investigated by UV/visible and fluorescence spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other transferrins, melanotransferrin binds only one Fe3+ ion per molecule. The binding properties of its N-terminal site are similar to other transferrins, but its C-terminal site does not bind iron at all. The differences can be related to specific amino acid changes in the C-terminal site.

Antigens, Neoplasm↗
PubMed1991

Preliminary crystallographic studies of copper(II)- and oxalate-substituted human lactoferrin.

As part of a comparative study on the binding of different metals and anions by human lactoferrin, we have prepared and crystallized: (1) dicupric lactoferrin with Cu2+ and carbonate in each site (Cu2Lf); and (2) a lactoferrin complex with Cu2+ and carbonate in one site, and Cu2+ and oxalate in the other (Cu2oxLf). Crystals of Cu2Lf are orthorhombic: a = 155.9, b = 97.0, c = 56.0 A, space-group P2(1)2(1)2(1); those of Cu2oxLf are also orthorhombici a = 155.9, b = 97.1, c = 56.2 A, space-group P2(1)2(1)2(1). Both are isomorphous with diferric human lactoferrin, Fe2Lf. Diffractometer data to 2.6 A and 2.5 A have been collected for Cu2Lf and Cu2oxLf, respectively. Difference maps show that the main effect of substitution of Cu2+ for Fe3+ is a small shift (0.5 to 1.0 A) in the metal position in each site. For Cu2oxLf the oxalate ion is found to be accommodated in the C-lobe, bound to copper in a bidentate mode, causing only small local changes, in the positions of adjacent Arg and Tyr side-chains.

Binding Sites↗
PubMed1991

Structure, function and flexibility of human lactoferrin.

X-ray structure analyses of four different forms of human lactoferrin (diferric, dicupric, an oxalate-substituted dicupric, and apo-lactoferrin), and of bovine diferric lactoferrin, have revealed various ways in which the protein structure adapts to different structural and functional states. Comparison of diferric and dicupric lactoferrins has shown that different metals can, through slight variations in the metal position, have different stereochemistries and anion coordination without any significant change in the protein structure. Substitution of oxalate for carbonate, as seen in the structure of a hybrid dicupric complex with oxalate in one site and carbonate in the other, shows that larger anions can be accommodated by small side-chain movements in the binding site. The multidomain nature of lactoferrin also allows rigid body movements. Comparison of human and bovine lactoferrins, and of these with rabbit serum transferrin, shows that the relative orientations of the two lobes in each molecule can vary; these variations may contribute to differences in their binding properties. The structure of apo-lactoferrin demonstrates the importance of large-scale domain movements for metal binding and release and suggests that in solution an equilibrium exists between open and closed forms, with the open form being the active binding species. These structural forms are shown to be similar to those seen for bacterial periplasmic binding proteins, and lead to a common model for the various steps in the binding process.

Animals↗
PubMed1991

Chemosensitivity testing of small cell lung cancer using the MTT assay.

A simple colorimetric test, the MTT assay, has been adapted for chemosensitivity testing of human small cell lung cancer cell lines, and fresh tumour samples. Optimal conditions for clinical chemosensitivity testing were determined using established SCLC lines. Nineteen different chemotherapeutic agents were tested, and sixteen of them were found to be cytotoxic in this assay system. The drug sensitivity of a panel of 16 SCLC cell lines was measured and compared. There was very little intraexperiment variation, but the interexperiment variation was significant. Cell lines which were derived from patients who had not received chemotherapy at the time the cell line was established were more sensitive (to all but one of the drugs) than lines derived from treated patients, and the differences were statistically significant for two of the drugs. One cell line, NCI-H209, which was derived from an untreated patient, stood out as being the most sensitive or among the most sensitive to all of the drugs tested. Another cell line, H69AR, which is a multidrug resistant subline of the cell line NCI-H69, was the most resistant to many of the natural product drugs tested. Multiple drug chemosensitivity testing was performed on eight fresh tumour samples from SCLC patients (five pleural effusions, one lymph node, and two primary tumours). It was possible to perform chemosensitivity testing on all of the clinical samples in which sufficient tumour cells were available. The drug sensitivity of the clinical samples was, in most cases, within the same range as for the cell lines. Since this assay is very rapid and simple to perform, it may have practical applications in clinical drug sensitivity testing of human tumours.

Antineoplastic Agents↗
PubMed1990

Apolactoferrin structure demonstrates ligand-induced conformational change in transferrins.

Proteins of the transferrin family, which contains serum transferrin and lactoferrin, control iron levels in higher animals through their very tight (Kapp approximately 10(20)) but reversible binding of iron. These bilobate molecules have two binding sites, one per lobe, each housing one Fe3+ and the synergistic CO3(2-) ion. Crystallographic studies of human lactoferrin and rabbit serum transferrin in their iron-bound forms have characterized their binding sites and protein structure. Physical studies show that a substantial conformational change accompanies iron binding and release. We have addressed this phenomenon through crystal structure analysis of human apolactoferrin at 2.8 A resolution. In this structure the N-lobe binding cleft is wide open, following a domain rotation of 53 degrees, mediated by the pivoting of two helices and flexing of two interdomain polypeptide strands. Remarkably, the C-lobe cleft is closed, but unliganded. These observations have implications for transferrin function and for binding proteins in general.

Animals↗
PubMed1989

Structure of human lactoferrin: crystallographic structure analysis and refinement at 2.8 A resolution.

The structure of human lactoferrin has been refined crystallographically at 2.8 A (1 A = 0.1 nm) resolution using restrained least squares methods. The starting model was derived from a 3.2 A map phased by multiple isomorphous replacement with solvent flattening. Rebuilding during refinement made extensive use of these experimental phases, in combination with phases calculated from the partial model. The present model, which includes 681 of the 691 amino acid residues, two Fe3+, and two CO3(2-), gives an R factor of 0.206 for 17,266 observed reflections between 10 and 2.8 A resolution, with a root-mean-square deviation from standard bond lengths of 0.03 A. As a result of the refinement, two single-residue insertions and one 13-residue deletion have been made in the amino acid sequence, and details of the secondary structure and tertiary interactions have been clarified. The two lobes of the molecule, representing the N-terminal and C-terminal halves, have very similar folding, with a root-mean-square deviation, after superposition, of 1.32 A for 285 out of 330 C alpha atoms; the only major differences being in surface loops. Each lobe is subdivided into two dissimilar alpha/beta domains, one based on a six-stranded mixed beta-sheet, the other on a five-stranded mixed beta-sheet, with the iron site in the interdomain cleft. The two iron sites appear identical at the present resolution. Each iron atom is coordinated to four protein ligands, 2 Tyr, 1 Asp, 1 His, and the specific Co3(2-), which appears to bind to iron in a bidentate mode. The anion occupies a pocket between the iron and two positively charged groups on the protein, an arginine side-chain and the N terminus of helix 5, and may serve to neutralize this positive charge prior to iron binding. A large internal cavity, beyond the Arg side-chain, may account for the binding of larger anions as substitutes for CO3(2-). Residues on the other side of the iron site, near the interdomain crossover strands could provide secondary anion binding sites, and may explain the greater acid-stability of iron binding by lactoferrin, compared with serum transferrin. Interdomain and interlobe interactions, the roles of charged side-chains, heavy-atom binding sites, and the construction of the metal site in relation to the binding of different metals are also discussed.

Amino Acid Sequence↗
PubMed1989

Preliminary crystallographic studies on human apo-lactoferrin in its native and deglycosylated forms.

Human apo-lactoferrin in both native and deglycosylated forms has been purified, and crystals obtained by dialysis against low ionic strength buffer solutions. The crystals of native apo-lactoferrin are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 222.0 A, b = 115.6 A, c = 77.8 A and have two protein molecules per asymmetric unit. Two crystal forms of deglycosylated apo-lactoferrin have been obtained. One is orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 152.1 A, b = 94.6 A, c = 55.8 A. The second is tetragonal, space group I4, with cell dimensions a = b = 189.4 A, c = 55.1 A. Both of the latter have only one molecule per asymmetric unit, and are suitable for high-resolution X-ray structure analysis.

Apoproteins↗
PubMed1987

Structure of human lactoferrin at 3.2-A resolution.

The three-dimensional structure of human milk lactoferrin, a member of the transferrin family, has been determined crystallographically at 3.2-A resolution. The molecule has two-fold internal homology. The N- and C-terminal halves form two separate globular lobes, connected by a short alpha-helix, and carry one iron-binding site each. Each lobe has the same folding, based on two domains of similar supersecondary structure, with the iron site at the domain interface. Each iron atom is coordinated by four protein ligands: two tyrosines, one histidine, and one aspartate. A probable CO3(2-) (or HCO3-) ion is suggested by the electron density, bound to iron and adjacent to an arginine side chain and a helix N terminus. The protein folding and location of the binding sites show marked similarities with those of other binding proteins, notably the sulfate-binding protein from Salmonella typhimurium.

Binding Sites↗
PubMed1986

Preliminary crystallographic studies on bovine lactoferrin.

The purification of bovine lactoferrin, its crystallization at low ionic strength, and preliminary X-ray crystallographic data are reported. The crystals, which grow from a two-phase system, are radiation-stable and suitable for a medium-resolution X-ray analysis. They are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 138.4 A, b = 87.1 A, c = 73.6 A, and one protein molecule in the asymmetric unit.

Animals↗