Active-site engineering of carbonic anhydrase and its application to biosensors.
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Animals↗
Biomedical subjects
J A Hunt
Publications and source records attributed to J A Hunt.
Popliteal lymph node metastasis from primary cutaneous melanoma.
AIMS: To document the incidence of popliteal lymph node involvement by metastatic melanoma and to consider the implications of this information for clinical management. METHODS: From the computerized database of the Sydney Melanoma Unit, all patients with primary melanomas located at or distal to the knee were identified and their records were examined. Experience with those patients who developed popliteal node metastases was then reviewed. RESULTS: Thirteen of 4262 patients (0.31%) with primary melanomas of the distal lower limb developed popliteal node metastases. Six of the 13 patients had previous, synchronous or subsequent groin node metastases. CONCLUSIONS: Popliteal lymph node involvement by metastatic melanoma is a rare event. The study results suggest only two indications for full popliteal node clearance-either a histologically positive sentinel node in the popliteal fossa or clinical evidence of metastatic disease in a popliteal node.
Adolescent↗
Initial experience of Integra in the treatment of post-burn anterior cervical neck contracture.
Bilaminate bioartificial skin substitutes such as Integra have been described as a temporary substitute for autograft in acute burns. The aim of this study was to assess their role in the treatment of hypertrophic scarring and anterior neck contracture in the reconstructive phase of burns surgery. Five patients underwent Integra grafting over a 12-month period, with one patient having the procedure performed on two separate occasions. One patient had a combination of hypertrophic scarring of the face and neck contracture, and two patients suffered from recurrent folliculitis in hypertrophic scars in the bearded area of the neck. Mean hospital stay was 24 days (range, 19-30 days). Mean follow-up was 48 months (range, 43-54 months). In all cases of neck contracture a superior cosmetic result was achieved. Recurrence of contracture of greater than 50% occurred within the follow-up period in all cases. Ineffective immobilisation of the graft and overgranulation were identified as contributing to contracture recurrence. Based on our initial experience bilaminate artificial skin substitutes offer an improved cosmetic result in cases of neck contracture and hypertrophic scarring, particularly in the bearded area of the neck. Recurrence of neck contracture remains a problem despite improved techniques of immobilisation. Long-term follow-up will be necessary to assess the applications of this technique fully.
Adult↗
Novel disulfide engineering in human carbonic anhydrase II using the PAIRWISE side-chain geometry database.
An analysis of the pairwise side-chain packing geometries of cysteine residues observed in high-resolution protein crystal structures indicates that cysteine pairs have pronounced orientational preferences due to the geometric constraints of disulfide bond formation. A potential function was generated from these observations and used to evaluate models for novel disulfide bonds in human carbonic anhydrase II (HCAII). Three double-cysteine variants of HCAII were purified and the effective concentrations of their thiol groups were determined by titrations with glutathione and dithiothreitol. The effects of the cysteine mutations on the native state structure and stability were characterized by circular dichroism, enzymatic activity, sulfonamide binding, and guanidine hydrochloride titration. These analyses indicate that the PAIRWISE potential is a good predictor of the strength of the disulfide bond itself, but the overall structural and thermodynamic effects on the protein are complicated by additional factors. In particular, the effects of cysteine substitutions on the native state and the stabilization of compact nonnative states by the disulfide can override any stabilizing effect of the cross-link.
Carbonic Anhydrases↗
Active scatter factor (HGF/SF) in proliferative vitreoretinal disease.
PURPOSE: Hepatocyte growth factor/scatter factor (HGF/SF) possesses mitogenic, motogenic, and morphogenic properties and has recently been implicated in various retinal diseases. The role of HGF/SF in proliferative vitreoretinal disease was investigated. METHODS: Sections of epiretinal membranes were stained immunohistochemically for cytokeratins, to identify HRPE cells, and for HGF/SF receptor (c-Met). Cultured HRPE cells were stained for c-Met and investigated for shape change in response to HGF/SF, by using image analysis. The dose-response relationship for HRPE cells to HGF/SF was investigated by a cell migration assay and the specificity of this response evaluated by a neutralization experiment. Subretinal fluid (SRF) and vitreous from patients with retinal detachment and proliferative vitreoretinopathy (PVR) plus vitreous from eyes obtained after death, eyes with macular hole, and eyes with proliferative diabetic retinopathy (PDR) were investigated for the presence of HGF/SF using an enzyme-linked immunosorbent assay (ELISA). HGF/SF activity was measured using an MDCK cell scatter assay. RESULTS: HRPE cells in epiretinal membranes and in culture expressed c-Met. Cultured HRPE cells responded to HGF/SF by an epithelial-to-mesenchymal shape change and by cell migration, a response that increased with increasing concentrations of HGF/SF. This response was reduced in the presence of neutralizing antibody. There was evidence of HGF/SF in increasing concentrations in more severe PVR and in PDR when measured by ELISA, and, conversely, there was evidence of correspondingly decreasing HGF/SF activity when measured by MDCK cell scatter assay in these diseases. CONCLUSIONS: HGF/SF is present in normal and pathologic vitreous. HRPE cells respond by shape change and cell migration to HGF/SF. Concentrations of HGF/SF increase in proliferative vitreoretinal disease and increase in turn with increased severity of the disease, but HGF/SF bioactivity decreases (consistent with activator depletion). These findings are consistent with the hypothesis that HGF/SF may play a role in the HRPE mesenchymal transformation that typifies PVR.
Adult↗
Research: a new era for nursing.
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Diffusion of Innovation↗
Activation-dependent proteolytic degradation of polymorphonuclear CD11b.
CD11b/CD18 is the principal integrin of polymorphonuclear (PMN) leucocytes and is involved in their adhesion, migration and phagocytosis. In quiescent cells, the receptor is stored in intracellular granules from where it is translocated to the cell surface in response to a variety of stimuli. In this study, we demonstrated that strong stimulation of PMNs not only leads to the upregulation of CD11b surface expression, but also to the subsequent time-dependent apparent loss of this receptor, as detected by fluorescence-activated cell sorting (FACS) using a monoclonal antibody (mAb) against an N-terminal CD11b epitope. This epitope loss was observed following either direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate (PMA) or after multiple receptor stimulation using a combination of the agonist N-formylmethionyl-leucyl-phenylalanine (FMLP) and the priming agents granulocyte macrophage-colony stimulating factor (GM-CSF) and platelet factor (PF) 4. However, upregulation following weak stimulation with FMLP alone was not followed by subsequent epitope loss of the receptor. The increases and subsequent decreases in CD11b expression induced by PMA were paralleled by an increase and a decrease in PMN adhesion to CD11b-specific ligands, fibrinogen and intercellular adhesion molecule (ICAM)-1. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis showed that this epitope loss of PMN CD11b was the result of proteolytic degradation of the N-terminal region of the molecule. The use of a range of proteinase inhibitors indicated that this CD11b degradation involves a cell-associated serine proteinase. This is the first demonstration of the proteolytic alteration of CD11b in response to strong PMN stimulation. Given the central role of CD11b/CD18 in all aspects of PMN function, this alteration of the CD11b molecule and its effect on PMN adhesion are probably of considerable pathophysiological importance.
Antibodies, Monoclonal↗
Effects of mesh modification on the structure of a mandrel-grown biosynthetic vascular prosthesis.
Mandrel-grown, mesh-reinforced vascular prostheses require adequate tissue coverage of the mesh for effective clinical function, particularly in low blood flow situations. Development of the ovine collagen-based Omniflowtrade mark vascular prosthesis has shown that the extent of this tissue cover is dependent on the interactions of the mandrel and the mesh with the sheep host. In the present study, the effects of chemical changes to the mesh have been examined. These data indicate that certain treatments of the mesh, particularly collagen or heparin, lead to increased tissue coverage while the number of sheep cells present and the ultrastructure of the resulting vessel remain unchanged.
Animals↗
In vivo evaluation of modified mandrel-grown vascular prostheses.
The Omniflowtrade mark Vascular Prosthesis (OVP) has been manufactured and extensively tested in animal and human trials. It has mechanical and biological qualities superior to synthetic and biological conduits, particularly in low flow conditions. For further development into the smaller diameter coronary prostheses, the inner luminal surface is of paramount importance. In a previous study this inner surface was modified to produce a more uniformly thicker nonundulating surface. In this study the mandrels of these modified OVPs were treated with either collagen or heparin; the OVPs were evaluated for patency, tissue integration and wound healing, and endothelialization using a dog model comparable to that used to evaluate the unmodified OVP. In all instances, each of the modified prostheses were fully patent and had no signs of any deleterious effects caused by these modifications; no thrombus or aneurysms were visible. The tissue response was rapid with excellent new host collagen deposition within the vessel wall and minimal inflammatory and foreign body giant cells. Endothelialization was noted at the earliest explant time point in central regions of the prostheses, albeit that the histological picture at this time point appeared to reflect a complex atypical intimal layer.
Animals↗
Metal binding specificity in carbonic anhydrase is influenced by conserved hydrophobic core residues.
The role of highly conserved aromatic residues surrounding the zinc binding site of human carbonic anhydrase II (CAII) in determining the metal ion binding specificity of this enzyme has been examined by mutagenesis. Residues F93, F95, and W97 are located along a beta-strand containing two residues that coordinate zinc, H94 and H96, and these aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitutions of these aromatic amino acids with smaller side chains enhance the copper affinity (up to 100-fold) while decreasing the affinity of both cobalt and zinc, thereby altering the metal binding specificity up to 10(4)-fold. Furthermore, the free energy of the stability of native CAII, determined by solvent-induced denaturation, correlates positively with increased hydrophobicity of the amino acids at positions 93, 95, and 97 as well as with cobalt and zinc affinity. Conversely, increased copper affinity correlates with decreased protein stability. Zinc specificity is therefore enhanced by formation of the native enzyme structure. These data suggest that the hydrophobic cluster in CAII is important for orienting the histidine residues to stabilize metals bound with a distorted tetrahedral geometry and to destabilize the trigonal bipyramidal geometry of bound copper. Knowledge of the structural factors that lead to high metal ion specificity will aid in the design of metal ion biosensors and de novo catalytic sites.
Amino Acids↗
Determination of the breakpoint of the common alpha-thalassaemia deletion in Filipinos in Hawaii.
The region of the crossover causing the Filipino type of alpha thalassaemia has been determined by examining similarity between the regions which had been indicated as involved in the crossover points by restriction mapping, using the published alpha-globin region DNA sequence. The crossover point was found in 21 base pairs between two Alu sequences using PCR primers flanking these Alu sequences. A simple PCR multiplex assay has been devised to detect heterozygotes and homozygotes.
Base Sequence↗
Hypoxia augments cytokine (transforming growth factor-beta (TGF-beta) and IL-1)-induced vascular endothelial growth factor secretion by human synovial fibroblasts.
Vascular endothelial growth factor (VEGF) is abundant in synovium and synovial fluids, where it probably contributes to vascular permeability and angiogenesis in arthritic joints. To investigate the probable sources of VEGF in synovium, we compared the ability of several cytokines (TGF-beta, platelet-derived growth factor (PDGF), IL-1, tumour necrosis factor (TNF), basic fibroblast growth factor (bFGF) that are associated with arthritis and angiogenesis, to stimulate secretion of VEGF protein by human synovial fibroblasts. TGF-beta was the strongest inducer of VEGF secretion; six times more VEGF was secreted when cells were stimulated by TGF-beta than when stimulated by PDGF or IL-1 for 24 h. TNF-alpha and bFGF did not stimulate any secretion of VEGF. The stimulatory effects of TGF-beta and IL-1 on VEGF secretion were additive. Hypoxic culture alone also stimulated VEGF secretion, but more importantly, hypoxic culture conditions doubled the rate of VEGF secretion stimulated by the cytokines TGF-beta and IL-1. When dermal and synovial fibroblasts were stimulated identically by hypoxia and cytokines (TGF-beta and IL-1), synovial fibroblasts secreted four times more VEGF than did dermal fibroblasts. Thus in rheumatoid arthritis, the capacity of synovial fibroblasts in the hypoxic environment to secrete large amounts of VEGF in response to cytokines such as TGF-beta probably contributes significantly to angiogenesis in the synovium.
Arthritis↗
Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.
The beta chemokine eotaxin is a potent eosinophil activator and chemoattractant. We examined immunohistochemically eotaxin protein expression in a range of normal rat tissues and in rat lung during Sephadex particle-induced pulmonary inflammation. The time course of eotaxin expression in lung at various time points after Sephadex administration was related to the appearance of eosinophils in the bronchoalveolar lavage fluid and tissue distribution of eotaxin receptor (CCR3) positive cells. Results showed that eotaxin protein was constitutively expressed by both lung airway epithelial cells and gut epithelial cells in normal tissues in the absence of inflammation. During Sephadex induced pulmonary inflammation, eotaxin expression increased in alveolar macrophages prior to the major increase in eosinophil numbers which reached a peak at 72 h. The pattern of eotaxin pulmonary expression and the location of CCR3 receptor positive cells suggest a chemoattractant gradient resulting in migration firstly into the tissue and subsequently through the airway epithelium into the airways. Treatment of rats with the glucocorticoid dexamethasone or the immunosuppressant cyclosporin A reduced eosinophil entry into lung tissue and airways but had no apparent effect on eotaxin expression in vivo, indicating that both these drugs inhibit eosinophil recruitment either by an eotaxin-independent mechanism, or by targetting factors that synergise with eotaxin, or an event post eotaxin expression.
Animals↗
A specialist nurse: an identified professional role or a personal agenda?
Specialist nurses have existed for many years. Initially denoting a nurse with extensive clinical experience, implicit within nursing's professional agenda for attaining 'specialist' status since the 1960s has been the requirement to achieve a high degree of 'specialist' knowledge through post-basic education. Despite the professional agenda, much confusion surrounding definitions of specialist nurses prevails. In recent years this confusion has been compounded in the United Kingdom by the introduction of advanced nurse practitioners alongside existing clinical nurse specialists. This paper suggests that health care professionals' perceptions of a 'specialist' are subjective, grounded in their own experiences. Drawing on a study which examined relationships between paediatric oncology outreach nurse specialists and other health care professionals, two types of personal agenda from which perceived 'specialist' status evolves are described: (1) 'needs-driven agendas', and (2) 'peer-driven agendas'. When 'specialist' status is accorded to paediatric oncology outreach nurse specialists, 'specialist' knowledge is derived from: formal qualifications, hands-on technical skills, previous 'specialist' work experience, in-depth medical knowledge and/or insight into families' dynamics. The relative contribution each of these makes towards constructing a 'specialist' depends on the experiences of individual health care professionals and the varying work locations and professional backgrounds of paediatric oncology outreach nurse specialists.
Career Mobility↗
Using computer-based systems for biomaterial evaluation.
Computer-based image analysis techniques can be combined with immunohistochemistry to harness the full potential of computer technology. They can be used to quantitatively and reproducibly analyse cellular responses to materials and can play a significant role in medical device development and material selection. The benefits and pitfalls of the techniques are examined.
Biocompatible Materials↗
Haemocompatiblity of controlled release glass.
There are many medical applications which benefit from the use of soluble biomaterials, including the sustained release of drugs over a precise period of time, or temporary conduits for controlling nerve regrowth. We have manufactured a series of phosphate-based controlled release glasses (CRGs) in which the solubility could be controlled by varying the concentration of CaO and Na2O. Fibres of the CRG containing iron and cerium were placed into direct contact with human neutrophils and macrophages in tissue culture for 2.5 and 24 h respectively and the responses analysed by scanning electron microscopy (SEM) and confocal microscopy. The supernatants were analysed for the cytokine IL-1beta by enzyme-linked immunosorbent assay (ELISA). Disks of CRG of various compositions were placed in contact with whole blood for 30 min and platelet adhesion assessed by SEM. Activation of platelets, granulocytes and complement were quantified by ELISA for beta-thromboglobulin, elastase and iC3b. Intrinsic coagulation activation was measured by timing the clotting of recalcified plasma. Only the cerium fibre inhibited IL-1beta release from macrophages. No platelet adhesion was observed to any disk composition. Three compositions containing MgO inhibited plasma clotting and showed an insignificant level of complement activation. This study has demonstrated the development of a number of compositions of CRG, which have great potential in a wide variety of biomedical applications.
Journal Article↗
Soft tissue response to glycerol-suspended controlled-release glass particulate.
Vesicoureteral reflux and urinary incontinence have previously been treated by various means including the endoscopic delivery of injectable bulking materials such as silicone micro-implants, PTFE implants, glass particles, fat and bovine collagen. These first three materials do not degrade and collagen requires frequently repeated injections in order to sustain the restored continence provided. Vesicoureteric reflux in children usually resolves independently before the age of five. Correction is required before this, because treatment by prophylactic antibiotics is frequently unsuccessful in preventing breakthrough infection. The ideal material for injection should have large particles to avoid migration, inject easily and controllably, be non-toxic and dissolve over the period of time by which time the kidney will be mature. Three different controlled-release glass (CRG) granule compositions have been prepared by Giltech Ltd, and suspended in a suitable carrier medium (in this case glycerol). The degradable glasses, which have two different size ranges of 200-300 and <53 microm, and three different solution rates, were injected intramuscularly into the dorso-lumbar region of rats. Histological analysis of cryostat cut section after time periods of 2 d, 4 and 9 wk, and 6 mon has been performed. Histology sections were stained for neutrophils and macrophages using enzyme histochemistry. ED1 (monocytes and immature macrophages), ED2 (mature tissue macrophages), CD4 (helper/inducer T-lymphocytes and macrophages), CD8 (suppressor/cytotoxic T-lymphocytes), Interleukin-1beta, IL-2 (activated T-lymphocytes), Major Histocompatibility Complex (MHC) class II (activated macrophages and activated B-lymphocytes), alpha-beta (T-lymphocytes) and CD45RA (B lymphocytes) antibodies have been used to stain immunohistochemically each sample. This study demonstrates that particulate, degrading glass is stimulating an inflammatory response in soft tissue at time periods up to 6 mon. It should be noted that very small particulate, fast degrading glass is leading to tissue necrosis and should not be considered further for these applications. However, larger particulate, slower degrading materials are demonstrating effective potential for stress incontinence applications.
Journal Article↗
Quantification of the bone-related mRNAs at the bone/prosthetic interface.
Aseptic loosening is one of the major reasons for failure of joint prostheses. The periprosthetic tissue has previously been described microscopically; however, little work has been devoted towards quantitating genes expressed by cells at the materials/tissue interface. This study aims to characterize the phenotypic expression of osteoblasts and test the feasibility of quantifying the level of gene expression in periprosthetic tissue sections by combining in situ hybridization and image analysis techniques. There are many factors to consider when quantifying mRNA, in that comparing labeling between different cDNA probes, these should have comparable length and base comparison. The probes should be labeled with the same specific activity, that is the amount of probe to label added is the same, both between different probes and between batches of the same probe. Chromagen color reactions are variable in that the color development is not always linear and more likely follows a sigmoidal curve. Samples should only be compared when it is known that the reaction has been in the linear range. The image analysis of such staining introduces further factors which should be considered and controlled. Color analysis is a very complex problem with respect to reproducibly analyzing histological sections. The brightness component of the image should be independent of the colors within the image, in conventional RGB (red, green and blue) signalling mode this is not possible, while when using HSI (hue saturation and intensity) mode this becomes possible, and factors like staining intensity and brightness of the image become much more accountable and controllable. With these factors identified, we consider that the quantitative image analysis approach does allow comparison of patterns of bone-related mRNAs and demonstrates differences in expression in these osteogenic factors depending on distance from the prosthesis, tissue type, patient and device.
Journal Article↗