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V A Smith

Publications and source records attributed to V A Smith.

At least 19 recordsLinked to original sources
PubMed2006

Tissue inhibitor of metalloproteinase-3 differentially binds to components of Bruch's membrane.

BACKGROUND: Sorsby's fundus dystrophy (SFD) is caused by mutations in tissue inhibitor of metalloproteinase (TIMP)-3 and, with the exception of early onset, is similar to age-related macular degeneration. The pathological features of this condition relate to the accumulation of TIMP-3 in Bruch's membrane. AIMS: To compare the extracellular membrane-binding characteristics of wild-type and four SFD-mutant TIMP-3s. METHODS: COS-7 cells were transfected with wild-type, Ser-181, Gly-167, Ser-156 and Tyr-168 SFD-mutant TIMP-3 cDNA. The TIMP-3 proteins subsequently synthesised were harvested, analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, semiquantified by ELISA and used in binding assays on the basis of the retention of the wild-type and SFD-mutant TIMP-3 proteins by components of Bruch's membrane. RESULTS: SFD-mutant TIMP-3s could not be distinguished from wild-type TIMP-3 by the extents to which they aggregated or adhered to type-I collagen, type-IV collagen, fibronectin, laminin, elastin, chondroitin sulphates A, B and C, and heparin sulphate. Of these macromolecules, the wild-type and SFD-mutant TIMP-3s exhibited greatest affinity for elastin and laminin. CONCLUSION: The similarity in the physical and extracellular membrane-binding characteristics of wild-type and SFD-mutant TIMP-3s indicates that these properties are not responsible for the difference in timing of onset of SFD and age-related macular degeneration.

Animals↗
PubMed2006

Keratoconus: matrix metalloproteinase-2 activation and TIMP modulation.

Keratoconus is an ocular condition that causes corneal thinning, cone formation and scarring. In view of a hypothesis that activated MMP-2 may initiate or facilitate disease progression, the MMP-2/TIMP systems of stromal cells derived from normal and keratoconic corneas have been compared. To achieve this, stromal cell cultures were established from normal, clear keratoconic (KCS-1) and scarred keratoconic (KCS-2) corneas. The secreted MMP-2 was assayed using [(3)H]Type IV collagen and analysed by zymography. Optimally maintained and nutrient deprived cells were subsequently incubated with [(3)H]lysine. The secreted radiolabelled macromolecules were separated and quantified. The results obtained indicated that optimally maintained KCS-1 stromal cells produced more MMP-2 than normal stromal cells but not TIMP. Nutrient deprivation induced MMP-2 activation and cell death. Surviving cells upregulated TIMP-1 synthesis and in this respect became similar to the KCS-2 stromal cells that did not excessively generate activated MMP-2 or die as a consequence of nutrient deprivation. From these results, it was concluded that KCS-1 stromal cells over-expressed MMP-2 without increasing TIMP production. This may facilitate MMP-2 activation in vivo and hence advance the keratoconic condition. KCS-2 cultures over-expressed both MMP-2 and TIMP-1. Because TIMP-1 inhibits MMP-2 activity and protects against cell death it may be of significance in initiating repair processes and curtailing keratoconus.

Cells, Cultured↗
PubMed2004

Doxycycline-a role in ocular surface repair.

BACKGROUND/AIMS: Doxycycline is a broad spectrum antibiotic that chelates metal ions and is frequently used as part of the treatment of ocular surface diseases. Its therapeutic value has been ascribed to an ability to inhibit matrix metalloproteinase (MMP) activity and both MMP and IL-1 synthesis. The aim of this study was to evaluate the role of doxycycline as an inhibitor of corneal MMPs and assess its contribution to ocular surface repair mechanisms. METHODS: Corneal epithelial cell and keratocyte cultures were grown to confluence and incubated with IL-1alpha, LPS, doxycycline, or doxycycline and LPS in serum free medium for 4 days. The cells were either harvested and assayed for caspase-3 activity or stained with either AE5 or antivimentin antibodies. Media samples were concentrated and assayed for MMP activity by zymography or using a fluorigenic substrate. ELISA was used to quantify IL-1alpha, MMPs -1,-2,-3,-9, and TIMPs -1 and -2. RESULTS: IL-1alpha and LPS had no effect on MMP/TIMP production by cultured corneal epithelial cells and keratocytes. Corneal MMP-2 inhibition by doxycycline was partially [Ca(2+)] dependent but irreversible. At the minimum inhibitory concentration, 100 micro m, doxycycline had no apparent effect on MMP and TIMP production, but ultimately caused the death of keratocytes and some of the epithelial cells that detached from their basement membrane. Caspase-3 activity was not detected in dead or dying keratocytes. The mechanism of cell death in cultured corneal epithelial cells was not caspase-3 related apoptosis as the activity of this enzyme, normally detectable, was lost. The epithelial cells that survived doxycycline treatment did not bind antivimentin antibody and compared with controls, reacted less with the AE5 antibody. They were probably transient amplifying cells. CONCLUSIONS: Doxycycline irreversibly inhibits corneal MMP-2 activity by chelating the metal ions that are catalytically and structurally essential. Corneal MMP/TIMP production in vitro is not modulated by IL-1alpha, LPS, or doxycycline. The therapeutic value of doxycycline may depend upon its effective concentration at the ocular surface and probably relates to its chelating properties.

Anti-Bacterial Agents↗
PubMed2002

A framework for integrating the songbird brain.

Biological systems by default involve complex components with complex relationships. To decipher how biological systems work, we assume that one needs to integrate information over multiple levels of complexity. The songbird vocal communication system is ideal for such integration due to many years of ethological investigation and a discreet dedicated brain network. Here we announce the beginnings of a songbird brain integrative project that involves high-throughput, molecular, anatomical, electrophysiological and behavioral levels of analysis. We first formed a rationale for inclusion of specific biological levels of analysis, then developed high-throughput molecular technologies on songbird brains, developed technologies for combined analysis of electrophysiological activity and gene regulation in awake behaving animals, and developed bioinformatic tools that predict causal interactions within and between biological levels of organization. This integrative brain project is fitting for the interdisciplinary approaches taken in the current songbird issue of the Journal of Comparative Physiology A and is expected to be conducive to deciphering how brains generate and perceive complex behaviors.

Animals↗
PubMed2001

Tear film MMP accumulation and corneal disease.

BACKGROUND/AIMS: Matrix metalloproteinases (MMPs) accumulate in the tears of patients with active peripheral ulcerative keratitis (PUK) but it is unknown whether these enzymes have a central role in disease progression. The aims of the present investigation were to determine the source of these enzymes and to ascertain whether their accumulation in tears is a phenomenon specific to PUK or a general feature of other anterior segment diseases. METHODS: The experimental samples were obtained from the culture media of conjunctival and corneal epithelial cells, from fractionated blood plasma and leucocytes of healthy subjects and patients with rheumatoid arthritis, and from the tears of healthy subjects and patients with a variety of anterior segment diseases. The MMPs of all samples were visualised by zymography and tear samples were assayed using nitrophenol acetate and an MMP-9 susceptible quenched fluorescent peptide as substrate. RESULTS: The major MMPs that accumulate in the tears of patients with rheumatoid arthritis with active ocular disease are MMP-9 and a species of M(r) 116,000. By comparing the zymographic activity profiles of the gelatinases present in the samples obtained, it was deduced that the main source of these MMPs was granulocytes. Their accumulation in tears was not unique to patients with PUK; detectable amounts of the enzymes also occurred in the tears of patients with keratoconus with associated atopic disease, patients undergoing treatment for herpetic eye disease, and patients with systemic and non-systemic dry eye disease. CONCLUSION: The MMPs that accumulate in tears are mainly derived from granulocytes. This may be effected by autoimmune diseases that involve ocular tissue or by ocular diseases that induce an inflammatory response.

Adolescent↗
PubMed1999

Localisation of connective tissue and inhibition of autofluorescence in the human optic nerve and nerve head using a modified picrosirius red technique and confocal microscopy.

The use of picrosirius red to localise connective tissue in thin tissue sections viewed by bright-field microscopy is well documented. Its use on thin tissue sections imaged by fluorescence confocal microscopy has also been reported. Here we describe modifications to published procedures that allow picrosirius red staining of thick 60-microm sections and their subsequent analysis by confocal microscopy. The use of phosphomolybdic acid pre-treatment was found to be essential for confocal analysis; in addition to preventing non-specific staining, it also quenched tissue autofluorescence. By incubating sections free-floating, pre-treating them with phosphomolybdic acid for 30 min and imaging them using an argon ion laser we were able to use confocal microscopy to image the entire depth of 60-microm human optic nerve and nerve head sections stained with picrosirius red. The application of this modified picrosirius red and confocal microscopy technique should be useful for analysing the three-dimensional structure of the optic nerve and other tissues with a similarly complex arrangement of connective tissue.

Adult↗
PubMed1999

Nonunion of the distal radius.

We report five cases of nonunion of the distal radius. There were three women and two men. The mean age was 44 years (range, 34-56). All five patients were heavy tobacco smokers and three had a history of alcohol abuse. In three patients, union of the distal radius was obtained. Two had a persistent nonunion which was salvaged with a total wrist fusion.

Adult↗
PubMed1999

Role of ocular matrix metalloproteinases in peripheral ulcerative keratitis.

AIM: Peripheral ulcerative keratitis (PUK) is an ocular manifestation of rheumatoid arthritis and other similar systemic diseases. The purpose of this inquiry was to investigate the involvement of matrix metalloproteinases (MMPs) in the induction and/or maintenance of PUK. METHODS: Substrate gel electrophoresis was used to characterise the MMP activities secreted by primary cultures of keratocytes derived from normal and perforated pathological corneal specimens, and those present in tears of normal subjects and patients with PUK. Substrate specificity and the in vivo activity status of the secreted MMPs was assessed by SDS-polyacrylamide gel electrophoresis of standard collagens incubated in the presence or absence of the various enzyme preparations. RESULTS: In addition to MMP-2 of M(r) 66,000, cultured keratocytes derived from perforated corneas of patients with PUK abnormally produce the MMP-2 of apparent M(r) 62,000. Other MMPs and in particular MMP-9 of M(r) 92,000, also occur in the tears of these patients. Their visualisation on substrate polyacrylamide gels correlated with clinical manifestations of disease activity; during periods of disease quiescence they were barely detectable. The steroid prednisolone, frequently used in systemic therapy, had no effect on the in vitro activity of MMP-2, or on its production by cultured corneal keratocytes. Although the in vitro activity of MMP-2 was inhibited by both Cu(2+) and Zn(2+), Cu(2+) apparently induced the keratocytes to produce activated enzyme and Zn(2+) irreversibly inhibited their production of MMP-2. CONCLUSION: Overexpression of corneal MMP-2 and tear film MMP-9 are characteristic features of patients with PUK and their activation may be a crucial facet of disease initiation or progression. Although effective in systemic therapy for PUK, prednisolone had no direct control over corneal MMP-2 production or activity. Zn(2+) on the other hand inhibited both MMP-2 production and MMP-2 activity and may, therefore, be of therapeutic value if suitably formulated and used in conjunction with systemic steroid treatment.

Copper↗
PubMed1995

Over-expression of a gelatinase A activity in keratoconus.

Keratoconus is an ocular disorder in which the central cornea becomes thin, conical and frequently scarred. We are exploring the possibility that this condition is induced and maintained by proteases that exist in the corneal matrix in an activated form. In this study, the activities of the proteases secreted in vitro and in vivo by keratocytes of normal, clear keratoconic, scarred keratoconic and traumatically scarred corneas have been compared and partially characterised. Data obtained by assaying acyl transferase activity showed that the matrix metalloproteinases account for a minimum of 95% of the total protease secreted by cultured keratocytes. Their summated specific activity was consistently and significantly higher in the culture medium of keratoconic keratocytes than in the medium of other keratocyte cultures. Analysis of the individual protease activities secreted by these corneal keratocytes in vitro and in vivo by SDS-gelatin polyacrylamide gel electrophoresis showed that a gelatinase of molecular weight 65,000 is the major protease secreted by normal keratocytes. Whereas clear keratoconic and traumatically scarred corneal keratocytes secrete an additional activity of molecular weight 61,000, scarred keratoconic corneal keratocytes generally produced little or none of this gelatinase activity. Both activities may be ascribed to gelatinase A, and although the 61,000 molecular weight form may be a significant feature of keratoconus, neither appears to be active as secreted.

Acyltransferases↗
PubMed1992

The Distribution of Gibberellins in Vegetative Tissues of Pisum sativum L. : I. Biological and Biochemical Consequences of the le Mutation.

The concentrations of endogenous gibberellin (GA) 1, 5, 8, 19, 20, and 29 in the component tissues of maturing tall (Le) and dwarf (le) pea (Pisum sativum) plants have been determined. The following conclusions were drawn from the data obtained: (a) GA(20) and its metabolites accumulate only in the growing regions of Le and le plants; (b) the le mutation is biochemically expressed in all immature tissues of the dwarf plants; (c) the quantitative composition of the GA metabolites in the various immature tissues is variable; (d) the total GA concentration in apical buds, unexpanded leaves, and tendrils is considerably higher than in GA(1)-responsive stem tissue; and (e) there is very little GA accumulation of the inactive 2beta-hydroxylated GAs (GA(8) and GA(29)) in either the mature vegetative tissues or the roots of pea plants.

Journal Article↗
PubMed1992

Gibberellin A(1) Biosynthesis in Pisum sativum L. : II. Biological and Biochemical Consequences of the le Mutation.

A comparative study of the metabolism of radiolabeled gibberellin (GA) 1, 19, and 20 in isolated vegetative tissues of isogenic Le and le pea (Pisum sativum) plants incubated in vitro with the appropriate GA substrate is described. The results of this study provide evidence that the enzymes involved in the latter stages of GA biosynthesis are spatially separated within the growing pea plant. Apical buds were not apparently involved in the production of bioactive GA(1) or its immediate precursors. The primary site of synthesis of GA(20) from GA(19) was immature leaflets and tendrils, and the synthesis of bioactive GA(1) and its inactive catabolite GA(8) occurred predominantly in stem tissue. GA(29), the inactive catabolite of GA(20), was produced to varying extents in all the tissues examined. Little or no difference was observed in the ability of corresponding Le and le tissues to metabolize radiolabeled GA(1), GA(19), or even GA(20). During a fixed period of 24 hours, stems of plants carrying the le mutation produced slightly more [(3)H]GA(1) (and [(3)H]GA(29)) than those of Le plants. It has been concluded that the le mutation does not lie within the gene encoding the GA(20) 3beta-hydroxylase protein.

Journal Article↗
PubMed1991

The endogenous gibberellins of dwarf mutants of lettuce.

The gibberellin (GA) content of E-1, a tall genotype of early flowering lettuce (Lactuca sativa L.), and of three selected GA-responsive dwarfs, dwf1, dwf2, and dwf2(1), has been determined using (13)C-labeled internal standards and gas chromatographymass spectrometry (GC-MS). In the shoots of the E-1 parent, GA(1), 3-epi-GA(1), GA(3), GA(5), GA(8), GA(19), GA(20), GA(29), and GA(53) were identified by full scan GC-MS and Kovats retention indices. Purification by immunoaffinity chromatography selective for 13-hydroxy GAs, was necessary for GA identification. Relative to the parent E-1, the concentrations of GA(1), GA(8), GA(20), and GA(29) in the shoots of dwf2 plants were reduced to about 10% and in shoots of dwf2(1) plants to less than 50%. In dwf1 the levels of GA(1), GA(8), and GA(29) were also reduced to less than 50% of the parent E-1, but the level of GA(20) was fivefold higher than in E-1. Plant height was correlated with the endogenous levels of GA(1) and GA(8).

Journal Article↗
PubMed1990

Partial Purification and Characterization of the Gibberellin A(20) 3beta-Hydroxylase from Seeds of Phaseolus vulgaris.

The GA(20) 3beta-hydroxylase present in immature seeds of Phaseolus vulgaris has been partially purified and characterized. The physical characteristics of the enzyme are similar to those of the GA 2beta-hydroxylases present in mature and immature seeds of Pisum sativum. It is acid-labile, hydrophobic, and of M(r) 45,000. The enzyme catalyzes the synthesis of GA(1), GA(5), and GA(29) from GA(20). Activity is dependent upon the presence of Fe(2+), ascorbate, 2-oxoglutarate, and oxygen. 2-Oxoglutarate does not function as a cosubstrate; in the presence of the enzyme, succinate is not a reaction product.

Journal Article↗
PubMed1990

Nursing student attrition and implications for pre-admission advisement.

The current shortage of nurses and the decreasing student applicant pool supports the need for institutions of higher education to examine the problem of nursing student attrition and possible solutions. This study surveyed 227 non-returning students to identify attrition factors. The results implicate problems with class scheduling, inadequate financial resources, and employment responsibilities. Twenty four nursing faculty members were additionally surveyed to identify factors. The faculty respondents target poor study skills as the primary problem, a finding not agreed on by the former students. These identified problem areas implicate inadequate preadmission advisement. Students should be made aware of the personal and financial costs of a nursing education prior to admission. Preadmission advising that provides an applicant with a more complete understanding of the nursing educational process may help prevent student attrition and the loss of new nurses to the profession.

Counseling↗
PubMed1989

An immunological approach to gibberellin purification and quantification.

Gibberellin (GA) specific, high-affinity monoclonal antibodies have been used to assay the GA content of various plant tissues and to purify selected GAs by immunoaffinity chromatography. These immunological techniques may not stand alone as a general method of GA analysis. The results of this study indicate, however, that in conjunction with gas chromatography-mass spectrometry for positive GA identification, radioimmunoassay and immunoaffinity chromatography are extremely powerful tools for purifying and quantifying GAs from plant tissues.

Journal Article↗