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Biomedical subjects

A F Clark

Publications and source records attributed to A F Clark.

At least 19 recordsLinked to original sources
PubMed2000

The similarity of protein expression in trabecular meshwork and lamina cribrosa: implications for glaucoma.

The purpose of the present investigation was to compare protein expression in various ocular cells and tissues including the human trabecular meshwork (TM) and the lamina cribrosa (LC). To conduct the comparisons, we primarily utilized autofluorography of one-dimensional (1D) and high resolution, two-dimensional (2D) polyacrylamide gels of proteins from radiolabelled tissues and cultured cells. Results from the investigations indicated that patterns of protein expression from TM and LC were the most similar among the ocular cells and tissues compared.Specifically, these autofluorographic ' fingerprints' indicated that proteins in TM and LC cultured cells and tissue were exceptionally similar (a) in band position and intensity (1D gels) and (b) in spot congruence (2D gels) as compared to other ocular cells and tissues. We conclude that the TM and the LC, two ocular tissues intimately linked to the pathogenesis of primary open-angle glaucoma, display remarkable similarity in protein expression. This finding may have implications for the molecular etiology of glaucoma.

Aged↗
PubMed2000

Inner city disadvantage and family functioning.

The co-existence of types of social, environmental and family disadvantage and domains of family dysfunction is explored through individual interviews and use of the Family Assessment Device in a cross-sectional study of 143 inner city families. The dependence upon social welfare by the family and the overall degree of disadvantage experienced both correlate significantly with all forms of family dysfunction. Marital disruption, poor physical care of the home or of the child(ren) and poor quality of mothering correlate significantly with difficulties both in role allocation and in affective involvement. Those families suffering more types of environmental disadvantage are more likely also to be dysfunctional in each domain of family life. Some possible mechanisms for these associations are highlighted and their implications for planning of effective interventions discussed.

Adolescent↗
PubMed2000

Localization of MYOC transcripts in human eye and optic nerve by in situ hybridization.

PURPOSE: To evaluate MYOC (myocilin) gene expression at the RNA level in normal intact human eyes and optic nerve using in situ hybridization. METHODS: Normal human eyes and optic nerves from donors 62 to 83 years of age with no history of glaucoma were fixed, embedded in paraffin, and sectioned. Sections were hybridized with (35)S-labeled sense and antisense riboprobes derived from a full-length MYOC cDNA. RESULTS: High levels of MYOC expression were observed throughout the trabecular meshwork as well as in the most anterior nonfiltering meshwork (Schwalbe's line), in the scleral spur, and in the endothelial lining of Schlemm's canal. MYOC transcripts were also detected in the anterior corneal stroma, in the ciliary muscle, beneath the anterior border of the iris, in the iris stroma, and in the sclera. Expression in the retrolaminar region of the optic nerve was present in the pial septa that divide the nerve fiber bundles, in the perivascular connective tissue surrounding the central retinal vessels, and in the dura mater, arachnoid, and pia mater of the meningeal sheath surrounding the optic nerve. CONCLUSIONS: MYOC gene expression in the trabecular meshwork, Schlemm's canal, scleral spur, and ciliary muscle indicates a structural or functional role for myocilin in the regulation of aqueous humor outflow that may influence intraocular pressure. MYOC expression in the optic nerve suggests that changes in the structural, metabolic, or neurotropic support of the optic nerve may influence its susceptibility to glaucomatous damage.

Aged↗
PubMed2000

Human trabecular meshwork cells secrete neurotrophins and express neurotrophin receptors (Trk).

PURPOSE: The purpose of this study was to compare the mRNA expression of neurotrophins (NTs) and NT receptors (Trk) in cultured human trabecular meshwork (HTM) cells and ex vivo HTM tissues, to immunolocalize both NT and Trk receptors in cultured HTM cells, and to demonstrate secretion of NTs by HTM cells. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of NT and Trk receptor mRNAs in early-passaged, cultured HTM cells from donors of several ages. RT-PCR was used on ex vivo HTM tissues from donors to compare and contrast mRNA expression with cell culture results. In addition, immunohistochemistry was used to localize the translated NT and low- (p75) and high- (Trk) affinity NT receptor proteins within cultured HTM cells and trabecular meshwork tissues. Last, enzyme-linked immunoassay (ELISA) was used to demonstrate secretion of NTs by HTM cells. RESULTS: Amplification products of the expected size for NTs were detected in both cultured HTM cells and ex vivo HTM tissues. Specifically identified were amplification products for the following NTs: NGF, BDNF, NT-3, and NT-4. Amplification products for the full-length Trk A and Trk C high-affinity receptor were observed, as well as truncated isoforms for Trk B and Trk C. No amplification products were produced for the full-length Trk B receptor nor for the low-affinity p75 receptor. Immunohistochemistry indicated that proteins for the various NTs and full-length and truncated Trk receptors were translated by cultured HTM cells and tissues. Immunoassays (ELISA) detected BDNF, NT-4, NGF, and NT-3 in the culture media from HTM cells. CONCLUSIONS: The results demonstrate, for the first time, mRNA expression for NT and Trk receptors by both cultured HTM cells and ex vivo HTM tissues. NTs were immunolocalized in HTM tissues and cultured HTM cells are capable of secreting NTs. Specific NTs acting through high-affinity Trk receptors within the HTM may be involved in maintaining the normal function of this complex tissue.

Adolescent↗
PubMed1999

Fas-activated apoptosis and apoptosis mediators in human trabecular meshwork cells.

A gradual loss of cells occurs within the human trabecular meshwork during normal aging and appears to be increased in patients with primary open-angle glaucoma. The exact mechanism by which cells are lost in either condition is not known, however phagocytosis, cell migration and cell death have been suggested. Apoptosis is one method by which cell death can occur. We have examined the modulators for apoptosis within the human trabecular meshwork using both cell lines and ex-vivo dissected trabecular meshwork tissues obtained from normal donors. Using RT-PCR it was shown that mRNA for several modulators of apoptosis (Fas, Bcl-2, Bcl-xl, Bax, and ICE) are expressed by both cell lines and ex-vivo tissues. Apoptosis was stimulated to occur by treating cell lines with a monoclonal antibody (IgM) to Fas. Apoptosis was verified via morphological changes to the cells, transferase-mediated dUTP nick-end labeling TUNEL Immunofluorescence, and DNA laddering. Control cells exposed to IgM did not undergo apoptosis. These results represent the first report of apoptosis modulators within the human trabecular meshwork and demonstrate that human trabecular meshwork cells can be stimulated to undergo apoptosis via the Fas/FasL pathway.

Aged↗
PubMed1999

Effects of glucocorticoids on the trabecular meshwork: towards a better understanding of glaucoma.

Glucocorticoid effects on the human trabecular meshwork can be used as a model system in which to study glaucomatous damage to the trabecular meshwork. One of the most important risk factors for glaucoma is an elevated intraocular pressure. The administration of glucocorticoids also can cause elevated intraocular pressure in some individuals. In addition, there is suggestive evidence linking glucocorticoids with the development of glaucoma. Glucocorticoids cause multiple effects on the human trabecular meshwork including changes in extracellular matrix metabolism, organisation of the cytoskeleton, and changes in gene expression and cell function. New discoveries on the molecular mechanisms of glucocorticoid receptor action provide new opportunities to study the possible role of this receptor in the development of glaucoma. For example, alternate spliced forms of the glucocorticoid receptor, glucocorticoid receptor response element half-sites, numerous modulatory factors, and direct effects of nuclear transcription factors have been recently described. Other recent information has shown that the new glaucoma gene (GLC1A/myocilin) is induced in the human trabecular meshwork by glucocorticoids. Although the exact function of myocilin is currently unknown, it offers the opportunity to dissect the molecular pathways regulating aqueous humor outflow. Future challenges include determining (1) which glucocorticoid effects in the human trabecular meshwork are responsible for elevated intraocular pressure; and (2) the significance of these findings to the development of glaucoma.

Animals↗
PubMed1999

Early graft function and patient survival following cadaveric renal transplantation.

BACKGROUND: The influence of events that occur early following renal transplantation such as delayed graft function (DGF) and acute rejection on long-term graft survival has been widely reported, but its association with patient survival has received less attention. METHODS: We studied 589 patients who received their first cadaveric transplants between 1984 and 1993, all of whom received cyclosporine-based immunosuppression and who had a median follow-up of seven years. The following factors were identified, and both univariate and multivariate analyses were used to determine their association with long-term patient and graft survival: age, sex, duration of pretransplant dialysis, primary renal disease, immediate graft function (IGF), DGF, primary nonfunction (PNF), acute rejection, and serum creatinine at 3, 6, and 12 months. RESULTS: Patients with PNF had a poorer survival than those with DGF and IGF (P = 0.01), but there was no difference in survival between DGF and IGF (P = 0.54). Good graft function (serum creatinine of less than 200 mumol/liter) at three months was predictive of better long-term patient survival (P = 0.03). Other factors associated with poor patient outcome were older age, diabetes, adult polycystic kidney disease, male gender, and acute rejection. Cardiovascular disease was the most common cause of death (51.8%). Good graft function at three months (P < 0.001) and an absence of rejection episodes (P = 0.01) were associated with better graft survival. CONCLUSION: Patients with poor levels of early graft function (but not DGF) and those with either acute rejection episodes or early graft loss are at an increased risk of early death. These high-risk groups should be targeted for interventional studies in an attempt to improve patient survival.

Adolescent↗
PubMed1999

Angiostatic activity of steroids in the chick embryo CAM and rabbit cornea models of neovascularization.

Ocular neovascular diseases represent a major cause of blindness in the world. Angiostatic steroids are a unique class of compounds which inhibit the formation of new blood vessels in various models, including ocular models of angiogenesis. In search of potent new anti-angiogenic agents for the treatment of ocular neovascular disease, a large group of steroids were evaluated for angiostatic activity in the chick embryo CAM model. Angiostatic activity was found among all steroid classes included in the study. There was a good correlation between the angiostatic efficacies of 15 diverse steroids tested in the chick CAM and in the rabbit LPS-induced corneal pocket models of neovascularization (r=0.76, p=0.01). These studies show that potent angiostatic steroids inhibit neovascularization in two different animal models, suggesting a common mechanism of action. Glucocorticoid therapy is sometimes associated with ocular side effects. Two of the most potent angiostatic steroids, AL-3789 and AL-4940, were evaluated for glucocorticoid-mediated antiinflammatory activity in the in vitro U937 cell model of LPS-induced IL-1 induction and found to be devoid of glucocorticoid activity. Angiostatic steroids which lack glucocorticoid activity should be attractive drug candidates for treating ocular neovascular disease.

Angiogenesis Inhibitors↗
PubMed1999

Age-related permeability changes in rabbit corneas.

The present study was designed to determine whether the corneal penetration of test compounds is altered in aging. Experiments were carried out by means of passive transport under steady-state conditions using in vitro diffusion cells. Permeabilities of a variety of compounds with different physicochemical properties were measured in young (six weeks) and old (three to four years) intact and denuded (wounded) rabbit corneas. There was a marked difference in penetration of compounds between young and aged corneas. A significant decrease in permeability with age was observed. The degree of difference depended on the lipophilicity and molecular weight of the compound and the integrity of the epithelial cell layer. The difference was more pronounced for large hydrophilic than small lipophilic compounds in the intact corneas. The difference in permeabilities of test compounds between young and old denuded corneas was essentially the same (about 2-fold). These studies provide clues to the fundamental biochemical difference in young and old corneas and better enables the development of rationales for efficient drug and nutrient delivery across the cornea.

Aging↗
PubMed1999

Inhibition of intraocular tumor growth by topical application of the angiostatic steroid anecortave acetate.

PURPOSE: This study examined the effect of an angiostatic agent on the growth of a highly vascularized intraocular tumor. METHODS: A murine uveal melanoma cell line (99E1) was transplanted intracamerally into athymic nude BALB/c mice. Mice were treated topically three times per day beginning on the day of tumor transplantation and continuing through day 28. Groups included (a) 1% anecortave acetate, (b) vehicle control, or (c) no treatment. Tumor growth was scored clinically according to the volume of anterior chamber occupied by tumor. Intraocular tumor weights were determined on days 10, 14, 21, and 28. The effect of the test agents on tumor cell proliferation was examined in vitro by [3H]thymidine incorporation. RESULTS: Tumors grew progressively in untreated mice and mice treated with the vehicle; tumors filled the entire eye by day 20 and frequently perforated the globe by day 21. By contrast, tumors treated with anecortave acetate grew significantly slower (P < 0.025) and did not perforate the eye. On days 21 and 28 the net tumor weight of the AL-3789-treated animals was 40% to 30% of controls (P < 0.05). Tumor inhibition was presumably due to the angiostatic properties of anecortave acetate because the compound did not affect tumor cell proliferation in vitro. CONCLUSIONS: The topical ocular administration of anecortave acetate restricted the growth of a highly vascularized angiogenic intraocular tumor.

Administration, Topical↗
PubMed1999

Expression of alternatively spliced growth factor receptor isoforms in the human trabecular meshwork.

PURPOSE: Growth factors act through high-affinity cell surface receptors expressed by target cells and are critical modulators of cell function. Because aqueous humor is known to contain growth factors, these molecules may play a key role in maintaining the normal function of the human trabecular meshwork (HTM). Alternate mRNA splicing is an important mechanism used by cells to generate diverse isoforms of growth factor receptors. Although previous investigators have suggested that HTM cells may express alternative isoforms of several growth factor receptors, there have been no studies to verify these preliminary findings. The objective of this study was to determine whether cultured and ex vivo HTM cells express alternate isoforms of hepatocyte, keratinocyte, and transforming growth factor beta (TGFbeta)-II receptors and to characterize the isoform molecular sequences. METHODS: To determine whether cells within the HTM express mRNA for alternate isoforms of growth factor receptors, total RNA was isolated from several well-characterized HTM cell lines that were established from donors of various ages and from fresh ex vivo HTM tissues from healthy donors. After cDNA synthesis, polymerase chain reaction was initiated using specific primers for alternate forms of the following receptors: hepatocyte growth factor (HGFR), keratinocyte growth factor (KGFR), and transforming growth factor beta receptor II (TGFbetaR-II). Specificity and characterization of the polymerase chain reaction amplification products were determined by nucleic acid sequencing. RESULTS: Amplification products of the expected size for the growth factor isoforms were expressed in cell lines and in ex vivo tissues. Nucleic acid sequencing showed that cultured HTM cells and fresh ex vivo trabecular meshwork tissues expressed specific mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II The HGFR alternate isoform contained a 96-bp insert in the C-terminal coding region of the cytoplasmic tyrosine kinase domain. The KGFR alternate isoform is a soluble, truncated form, because it has no transmembrane or cytoplasmic domain as does the normal membrane-associated form. The TGFbetaR-II alternate isoform contained a 75-bp insert in the N-terminal coding region of the extracellular domain. CONCLUSIONS: In vitro and ex vivo HTM cells express mRNA for alternatively spliced isoforms of HGFR, KGFR, and TGFbetaR-II. These alternatively spliced receptor isoforms may be functional within the HTM and may play a critical role in maintaining the normal microenvironment of this important tissue.

Aged↗
PubMed1998

The effect of dexamethasone on integrin and laminin expression in cultured human trabecular meshwork cells.

Glucocorticoid treatment in vivo can produce a glaucoma similar in many ways to POAG. Treatment of trabecular meshwork cells in culture with dexamethasone allows the study of biochemical aspects of this disease process. The effects of dexamethasone on the expression of integrins and laminin in both normal and glaucomatous cultured human trabecular meshwork cells were evaluated. Human trabecular meshwork cell lines were cultured for 18 days in the presence or absence of 10(-7) m dexamethasone. Radioimmunoprecipitation was used to determine the relative expression of five alphaintegrin subunits. Laminin expression was evaluated with Western blots. Laminin was increased in all cell lines following dexamethasone treatment. alpha2, alpha5 and alphaV integrin chains showed consistent dexamethasone-induced changes in expression, while alpha3 and alpha4 subunits did not. There were no differences in the expression patterns for any of these integrin subunits between normal and glaucomatous cell lines. Increased laminin deposition as seen in this study with dexamethasone treatment may be partially responsible for the decreased outflow facility seen in both steroid-induced glaucoma and in POAG.

Blotting, Western↗
PubMed1998

Cultured human trabecular meshwork cells express functional growth factor receptors.

PURPOSE: To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors. METHODS: The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors. RESULTS: Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification. CONCLUSIONS: These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Adolescent↗
PubMed1998

Treatment of schizophrenia in childhood and adolescence.

This paper reviews the management of schizophrenia occurring during childhood and adolescence. It considers the clinical features of the disorder particular to its early onset before providing a practical framework for assessment and treatment based upon a critical review of the available literature. A multi-modal approach to treatment encompassing the individual and their family is adopted with the roles of pharmacological, psychological, and environmental interventions all considered. The place of the newer "atypical" antipsychotic agents and the likelihood that they will soon become the first-line drugs of choice is particularly discussed.

Adolescent↗
PubMed1997

Renin-1 is essential for normal renal juxtaglomerular cell granulation and macula densa morphology.

The secretion of renin from granules stored in renal juxtaglomerular cells plays a key role in blood pressure homeostasis. The synthesis and release of renin and the extent of granulation is regulated by several mechanisms including signaling from the macula densa, neuronal input, and blood pressure. Through the use of a gene-targeting vector containing homology arms generated using the polymerase chain reaction, we have inactivated the Ren-1(d) gene, one of two mouse genes encoding renin, and report that lack of renin-1(d) results in altered morphology of the macula densa of the kidney distal tubule and complete absence of juxtaglomerular cell granulation. Furthermore, Ren-1(d-/-) mice exhibit sexually dimorphic hypotension. The altered growth morphology of the macula densa in Ren-1(d)-null mice should provide a tool for the investigation of the JG cell-macula densa signaling. Furthermore, the current data indicate that expression of the Ren-1(d) gene is a prerequisite for the formation of storage granules, even though the related protein renin-2 is present in these mice, suggesting that renin-1(d) and renin-2 are secreted by distinct pathways in vivo.

Animals↗
PubMed1997

Identification of a gene that causes primary open angle glaucoma.

Glaucoma is a major cause of blindness and is characterized by progressive degeneration of the optic nerve and is usually associated with elevated intraocular pressure. Analyses of sequence tagged site (STS) content and haplotype sharing between families affected with chromosome 1q-linked open angle glaucoma (GLC1A) were used to prioritize candidate genes for mutation screening. A gene encoding a trabecular meshwork protein (TIGR) mapped to the narrowest disease interval by STS content and radiation hybrid mapping. Thirteen glaucoma patients were found to have one of three mutations in this gene (3.9 percent of the population studied). One of these mutations was also found in a control individual (0.2 percent). Identification of these mutations will aid in early diagnosis, which is essential for optimal application of existing therapies.

Base Sequence↗
PubMed1997

Steroid-induced cataract: new perspective from in vitro and lens culture studies.

The prevailing view regarding the mechanism of steroid cataract formation holds that glucocorticoids are covalently bound to lens proteins resulting in destabilization of the protein structure allowing further modification (i.e. oxidation) leading to cataract. Alternative hypotheses (e.g. that cataracts result from glucocorticoid receptor mediated effects) have been difficult to test since protein binding does in fact occur for many cataractogenic steroids. A glucocorticoid lacking the typical glucocorticoid hydroxy group at C21 (fluorometholone, FML), other steroids which can bind to proteins but lack glucocorticoid activity, and a glucocorticoid antagonist (RU486) have been utilized to discriminate between these two hypotheses. Purified bovine beta-crystallin incubated with three different 3H-steroids, dexamethasone (Dex), aldosterone or progesterone demonstrated that the C-21 hydroxyl group is not essential for steroid binding. Progesterone (with no C-21 OH) bound to the greatest extent. Pretreatment of the protein with aspirin to acetylate the free protein amino groups blocked this binding, demonstrating the probability of a Schiff base mechanism. Lens culture studies with the same three radiolabeled steroids demonstrated much the same result. Rat lenses cultured for 48 hr-11 days, demonstrated that loss of GSH is an early and significant effect of several glucocorticoids (Dex, prednisolone and FML) but is not seen with other non-glucocorticoid steroids. However, none of the steroids tested consistently produced lenticular opacification (i.e. cataracts) in this in vitro system, nor did they alter rubidium transport. We suggest that a mechanism other than covalent binding of steroids to lens proteins is responsible for glucocorticoid induced cataracts because: (1) non-glucocorticoids were demonstrated to bind lens proteins as well or better than the glucocorticoid Dex and (2) only glucocorticoids, and not other steroids, lowered lens reduced glutathione content which has been demonstrated to be associated with other forms of cataract.

Aldosterone↗