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Biomedical subjects

J A McDonald

Publications and source records attributed to J A McDonald.

At least 19 recordsLinked to original sources
PubMed1996

Breaking the integrin hinge. A defined structural constraint regulates integrin signaling.

Integrins are heterodimeric (alpha, beta) cell adhesion receptors. We demonstrate that point mutations in the cytoplasmic domains of both the alpha and beta subunits promote constitutive signaling by the integrin alphaIIbbeta3. By generating charge reversal mutations, we show these "activating" mutations may act by disrupting a potential salt bridge between the membrane-proximal portions of the alpha and beta subunit cytoplasmic domains. Thus, the modulation of specific interactions between the alpha and beta subunit cytoplasmic domains may regulate transmembrane signaling through integrins. In addition, these activating mutations induce dominant alterations in cellular behavior, such as the assembly of the extracellular matrix. Consequently, somatic mutations in integrin cytoplasmic domains could have profound effects in vivo on integrin-dependent functions such as matrix assembly, cell migration, and anchorage-dependent cell growth and survival.

Amino Acid Sequence↗
PubMed1995

Integrin activation and cytoskeletal interaction are essential for the assembly of a fibronectin matrix.

Fibronectin (Fn) matrices are vital to vertebrate development and wound healing and modulate tumorigenesis. We used a recombinant Fn-binding integrin alpha IIb beta3, to define rules for integrin-initiated Fn matrix formation. We report the following. First, multiple Fn-binding integrins can support matrix assembly; their activation state controls fibrillogenesis. Second, Fn binding to cells expressing an activated integrin is necessary but not sufficient for matrix assembly. Additional "postoccupancy" events involving the integrin beta, but not the alpha subunit, cytoplasmic domain are needed. Third, these postoccupancy events require an intact actin cytoskeleton. We propose a model for integrin involvement in Fn fibrillogenesis that reconciles previous paradoxes and suggests novel approaches to the therapeutic control of Fn matrix assembly.

Actins↗
PubMed1995

The human and mouse receptors for hyaluronan-mediated motility, RHAMM, genes (HMMR) map to human chromosome 5q33.2-qter and mouse chromosome 11.

The gene for the receptor for hyaluronan-mediated motility, RHAMM (designated hyaluronan-mediated motility receptor, HMMR (human) and Hmmr (mouse), for mapping purposes), was localized to human chromosome 5q33.2-qter by somatic cell and radiation hybrid analyses. Investigation of two interspecific backcrosses localized the mouse RHAMM (Hmmr) locus 18 cM from the centromere of mouse chromosome 11 within a region of synteny homology with human chromosome 5q23-q35 genes. The map position of the human RHAMM gene places it in a region comparatively rich in disease-associated genes, including those for low-frequency hearing loss, dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q- syndrome. The RHAMM gene location and its ability to transform cells when overexpressed implicate RHAMM as a possible candidate gene in the pathogenesis of the recently described t(5;14)(q33-q34;q11) acute lymphoblastic leukemias.

Animals↗
PubMed1995

Expression of the integrin subunit alpha 9 in the murine embryo.

The alpha 9 integrin subunit is expressed in adult skeletal muscle, visceral smooth muscle, hepatocytes, squamous epithelium, and airway epithelium. The in vivo function of this protein is unknown. Thus far, only a single alpha 9-containing integrin has been identified (alpha 9 beta 1) and only a single ligand (tenascin) has been found for this integrin. In order to gain insight into the potential function of alpha 9 integrin(s), we examined the spatiotemporal distribution of the alpha 9 subunit and tenascin during murine embryogenesis. In all tissues where alpha 9 was expressed, its appearance was associated with other evidence of cell differentiation. In developing airway, visceral, and vascular smooth muscles, the onset of alpha 9 expression either coincided with or immediately followed the expression of alpha-SM actin. Expression of alpha 9 in epithelia was restricted to the choroid plexus and the basal cell layer of squamous epithelia where its appearance coincided with the development of stratification. alpha 9 immunostaining was first detected in developing skeletal musculature when skeletal myotubes formed. Tenascin expression was detected in many, but not all tissues found to express alpha 9. For example, the hair germs of maturing hair follicles exhibited high levels of alpha 9 staining, but no tenascin immunoreactivity was detected either within the hair germ themselves or in the adjacent dermis. In some tissues where tenascin expression colocalized with alpha 9, expression patterns were not synchronous. Although alpha 9 expression was associated with the onset of tissue differentiation, its expression was not limited to terminally differentiated cells. In fact, in the skin, alpha 9 expression appeared restricted to cells known to retain the capacity to proliferate, i.e., basal cells and hair germs. Thus, alpha 9 integrin(s) are not likely to contribute to the early steps in organ formation, but probably play a role in the maturation and/or maintenance of a variety of differentiated tissues. The expression of alpha 9 without its only known ligand, tenascin, suggests the existence of additional ligands.

Animals↗
PubMed1995

Neuronal production of fibronectin in the cerebral cortex during migration and layer formation is unique to specific cortical domains.

The distribution of fibronectin (FN) changes rapidly during early development of the cerebral cortex, but its cellular source is not known. With in situ hybridization we find two spatially and temporally distinct periods of FN mRNA expression in the embryonic and early postnatal cortex of the mouse. Before and during formation of the preplate by the first postmitotic neurons, FN mRNA levels are high throughout the telencephalic vesicle, deep in the neuroepithelial proliferative zone that contains dividing cells and the cell bodies of radial glia; expression in the cortical proliferative zone is limited to the period of neurogenesis. Just after the cortical plate is formed within the preplate, FN mRNA is expressed in the intermediate zone, which contains migrating neurons, and in the cortical plate, where neurons migrate past their predecessors to form layers. Brefeldin A treatment of an organotypic slice preparation demonstrates FN production in the intermediate zone and cortical plate, in locations that correspond exactly to the distribution of FN mRNA by in situ hybridization. FN-producing cells immunolabel with neuron-specific markers; in the intermediate zone and lower cortical plate they have morphological features characteristic of migrating neurons and are closely apposed to radial glia. FN mRNA expression and protein production continue in neurons of the cortical plate through the period of layer formation and then are downregulated. Examination of dissociated cortical cells by laser confocal microscopy confirms that FN accumulation after brefeldin A treatment is intracellular in neurons as well as in glia. Neuroepithelial expression of FN mRNA takes place throughout the telencephalon; FN produced by neurons is restricted to cells migrating toward and into specific cortical domains that include neocortex, insular and perirhinal cortex, and subiculum. Thus FN may be involved initially in supporting the cell division and fate determination that takes place in the neuroepithelium; later production by migrating neurons may play a role in the selection of radial glial pathways that lead to specific cortical regions, and in interactions between neurons as they form cortical layers within these regions.

Animals↗
PubMed1995

Growth factor and procollagen type I gene expression in human liver disease.

BACKGROUND/AIMS: Growth factors have been implicated in the pathogenesis of liver fibrosis, a major determinant of the clinical course of chronic liver disease. The aim of this study was to study the relationship of growth factor expression to inflammation and fibrosis in a variety of human liver diseases. METHODS: We studied by in situ hybridization the expression of transforming growth factor (TGF) beta 1, platelet-derived growth factor (PDGF) A and PDGF-B, and procollagen type I (pro-I) messenger RNAs (mRNAs) in liver diseases of various etiologies. RESULTS: Pro-I mRNA was expressed by mesenchymal cells at sites of inflammation and scarring, where TGF-beta 1 immunoreactivity was often found, and by perisinusoidal cells. TGF-beta 1 and PDGF-A mRNAs were expressed mainly by mononuclear cells and proliferating ductular cells. TGF-beta 1 mRNA was also expressed by perisinusoidal cells. PDGF-A gene expression was more common than that of PDGF-B. Pro-I and TGF-beta 1 expression correlated with both ductular proliferation and tissue inflammation, whereas PDGF-A and PDGF-B only correlated with ductular proliferation. CONCLUSIONS: Our data suggest that TGF-beta 1 and PDGF are involved in human liver inflammation and fibrosis. The expression of growth factor mRNAs in proliferating ductular cells may indicate a role for these cells in liver fibrogenesis and may help explain the pathophysiology of conditions such as biliary atresia progressing to fibrosis despite the absence of marked inflammation.

Gene Expression↗
PubMed1995

Hypothalamic-pituitary-testicular function in end-stage non-alcoholic liver disease before and after liver transplantation.

OBJECTIVE: Gonadal dysfunction is common in chronic liver disease, but most of the previous studies have been restricted to men with alcohol-induced liver disease. We have evaluated hypothalamic-pituitary-testicular function in patients with end-stage non-alcoholic liver disease before and at 6 and 12 months after hepatic transplantation. DESIGN: A prospective study of hypothalamic-pituitary-testicular endocrine function before and after cadaveric hepatic transplantation. PATIENTS: Fifty four consecutive patients with end-stage, non-alcoholic liver disease were evaluated before and after liver transplantation. MEASUREMENTS: Hypothalamic-pituitary-testicular (HPT) axis function was evaluated under basal conditions by single morning measurements of plasma total and free testosterone, sex hormone-binding globulin and by plasma LH and FSH responses to 100 micrograms i.v. GnRH. RESULTS: Men with chronic non-alcoholic liver disease had reduced levels of total and free testosterone and increased levels of SHBG compared with controls with normal liver function. Total and free testosterone were positively correlated with basal and stimulated LH (but not FSH) concentrations. Gonadotrophin responses to GnRH were preserved but delayed compared with healthy controls consistent with a predominantly hypothalamic defect in regulation of pituitary-testicular function. Increasing severity of underlying liver disease was associated with declining total and free testosterone as well as peak GnRH-stimulated LH concentrations. Spironolactone treatment was associated with decreased circulating testosterone levels only in men with liver disease of intermediate severity (Child-Pugh class B). Following hepatic transplantation, total and free testosterone and SHBG concentrations returned progressively towards eugonadal control levels over the first 12 months but total and free testosterone levels remained subnormal. CONCLUSIONS: Hypothalamic-pituitary regulation of testicular function is impaired in end-stage non-alcoholic liver disease in proportion to the severity of underlying liver disease. Spironolactone reduces circulating testosterone but only among men with Child-Pugh B liver cirrhosis. Gonadal function improves, but is not normalized, over the first year following successful liver transplantation.

Adolescent↗
PubMed1995

The role of alpha 4 beta 1 integrin in cell motility and fibronectin matrix assembly.

The alpha 4 beta 1 integrin has been suggested to play important roles in embryogenesis and pathogenesis of many diseases which involve both cell adhesion and cell migration. Previous studies using anti-alpha 4 beta 1 antibodies and fibronectin (Fn) fragments have suggested that alpha 4 beta 1 integrins may be involved in cell motility on Fn and vascular cell adhesion molecule-1 (VCAM-1). However, the cells used in these studies also express other Fn integrin receptors including alpha 5 beta 1 integrin, which is known to function in cell motility on Fn. To test whether alpha 4 beta 1 integrins mediate cell motility on Fn and VCAM-1 in the absence of alpha 5 beta 1 integrin, we expressed human alpha 4 integrin in a Chinese hamster ovary (CHO) cell line that is deficient in alpha 5 beta 1 integrin (CHO B2). The parental alpha 5 deficient CHO B2 cells were unable to adhere, spread or migrate on Fn, nor could they assemble a fibrillar Fn matrix. Expression of alpha 4 beta 1 integrin in the CHO B2 cells enabled the cells to adhere, spread and migrate on Fn and on VCAM-1 but not to assemble a fibrillar Fn matrix. The cellular processes mediated by the interaction of alpha 4 beta 1 with Fn or VCAM-1 were inhibited by the CS1 peptide derived from the major alpha 4 beta 1 binding site on Fn. These findings demonstrate that alpha 4 beta 1 integrins not only function as cell adhesion receptors but also as cell motility receptors for Fn and VCAM-1 independent of alpha 5 beta 1. Moreover, they reveal important functional differences between Fn binding integrins. The alpha 4-positive, alpha 5-negative CHO cells described in this report will be useful tools in studying the mechanism of molecular signalling during integrin mediated cellular processes.

Amino Acid Sequence↗
PubMed1995

A novel role for alpha 3 beta 1 integrins in extracellular matrix assembly.

To study the biological role of alpha 3 beta 1 integrins in cell adhesion, migration, and in the deposition of extracellular matrix, we stably expressed the human alpha 3 integrin subunit in the alpha 4, alpha 5 integrin deficient CHO cell line B2. The expression of alpha 3 beta 1 integrins enhanced cell adhesion on entactin (also known as nidogen), but not on fibronectin. Using recombinant GST-fusion proteins that span the entire length of the entactin molecule, we located cell adhesive activity to the G2 domain of entactin. These results suggest that the alpha 3 beta 1 integrin functions as an adhesion receptor interacting with the G2 domain of entactin. On the other hand, the expression of alpha 3 beta 1 integrins did not confer the ability to migrate on entactin. Strikingly, the expression of alpha 3 beta 1 dramatically increased the deposition of entactin and fibronectin into the pericellular matrix. This was accompanied by increased binding activity of the 29 kDa amino-terminal domain of fibronectin. Thus, similar to alpha 5 beta 1 integrins, alpha 3 beta 1 integrins can play an important role in modulating the assembly of pericellular matrices. However, unlike fibronectin deposition supported by alpha 5 beta 1, alpha 3 beta 1 supported fibronectin deposition into pericellular matrix was not inhibited by antibodies binding to the RGD containing cell adhesion domain of fibronectin, demonstrating that the two processes are mechanistically distinct. The role of alpha 3 beta 1 in pericellular matrix assembly potentially implicates this receptor in the assembly and/or recognition of entactin-containing pericellular matrices, an observation consistent with its apparent role in the renal glomerulus of the mammalian kidney.

Amino Acid Sequence↗
PubMed1994

MSI brain scan machines, the grandfather of biosensors.

We all know about MRIs. You hurt your knee and for $800 the doctor can have a close look inside, from the outside, and the resulting images can often prevent costly and painful knee surgery. A similar technological breakthrough has been achieved for use in diagnosing brain disorders such as epilepsy. Instead of electrode insertion into one's brain at a cost of $50,000, Biomagnetic Technologies' (BTi) totally passive Magnetic Source Imaging (MSI) machines can detect incredibly subtle differences, and provide the critical information for 10% of that cost.

Amplifiers, Electronic↗
PubMed1994

Characterization of integrins in cultured human renal cortical tubule epithelial cells.

We have characterized the integrins present on cultured tubule epithelial cells from human renal cortexes, enriched for proximal cells, using fluorescence microscopy, immunoprecipitation, and cell adhesion assays. By immunofluorescence, the alpha 3-integrin subunit stained most intensely and was present on all cells predominantly at cell-cell contacts. The alpha 6-subunit was present on all cells in a pattern consistent with extracellular matrix contacts. The alpha 5-subunit was present on most cells in a cell-matrix contact pattern; alpha V-subunit was weakly positive and occasionally seen in cell-matrix contacts. The alpha 2-subunit was present on clusters of distal tubule cells, predominantly at cell-cell contacts. Immunoprecipitation revealed the predominant integrin to be alpha 3 beta 1 with some alpha 2 beta 1, presumably contributed by distal cells. The alpha 5 beta 1-, alpha 6 beta 1-, alpha 6 beta 4-, and alpha V beta 3-integrins, as well as trace amounts of alpha 1 beta 1-integrins, were also present. The alpha 4 beta 1-integrin was not detected. Initial attachment to fibronectin was mediated by alpha V beta 3- and alpha 5 beta 1-integrins; initial attachment to laminin was mediated by the alpha 6 beta 1- and alpha 3 beta 1- integrins and, in some preparations, by an unidentified integrin; and initial attachment to collagen type IV was mediated by alpha V beta 3-integrin and an unidentified beta 1-integrin. After extensively immunodepleting membrane extracts with anti-alpha 1, -alpha 2, -alpha 3, -alpha 4, -alpha 5, -alpha 6, and -alpha V antibodies, an anti-beta 1 antibody still precipitated an integrin. Its electrophoretic mobility differs from the laminin-binding alpha 7 beta 1-integrin. Thus we have identified many of the integrins on cortical tubule cells and their role in mediating initial attachment to extracellular matrix. However, the cell adhesion assays and immunoprecipitations suggest the presence of an unidentified beta 1-integrin that may mediate renal tubule cell attachment to laminin and collagen.

Cell Adhesion↗
PubMed1993

The alpha 5 beta 1 integrin fibronectin receptor, but not the alpha 5 cytoplasmic domain, functions in an early and essential step in fibronectin matrix assembly.

The alpha 5 beta 1 integrin mediates cell adhesion and migration on fibronectin, a glycoprotein critical for normal vertebrate embryonic development. Indirect evidence reported to date suggests that this receptor also functions in the deposition of fibronectin matrices. We used a molecular genetic approach to critically evaluate this role of alpha 5 beta 1 integrins. Mutant Chinese hamster ovary (CHO) cells deficient in alpha 5 integrin expression could not assemble a fibronectin matrix. Reconstituting alpha 5 beta 1 integrin expression by transfecting them with a full-length cDNA encoding the human alpha 5 chain completely restored fibronectin matrix assembly. CHO cells expressing an alpha 5 chain lacking the cytoplasmic domain also assembled a fibronectin matrix. Removing the cytoplasmic domain of alpha 5 appears to increase its activity in fibronectin matrix assembly. In addition to alpha 5 beta 1 integrin binding to fibronectin's RGD-containing domain, cells must bind with high affinity to fibronectin's amino-terminal 29-kDa matrix assembly domain to form a fibronectin matrix. Studies with the alpha 5-deficient CHO cells show that the expression of alpha 5 beta 1 integrin is also necessary for cells to bind fragments containing this distinct site in fibronectin and that a fibronectin matrix increases binding of the 29-kDa fragment. Thus, alpha 5 beta 1 integrins not only mediate cell adhesion to fibronectin, but also play an essential role in the assembly of a fibronectin matrix. This role includes direct binding to fibronectin and modulating a distinct binding event involving the interaction of fibronectin's amino-terminal matrix assembly domain with the cell surface.

Amino Acid Sequence↗
PubMed1993

Spire Corporation: a paradigm for success in the 90s.

There is much talk now of "defense conversion" and the need to turn a given country's "peace dividend" into improving its "infrastructure." Beyond the obvious political rhetoric, those terms bode well for the field of biotechnology. Every facet of technology must attend to its "delivery system," for the same obvious reason, roads and bridges must be maintained and improved. In the case of biosensors and their delivery into the human body, for example, it stands to reason that adequate focus should be paid to advancements in the field of the biomaterials from which they are made, or that house or carry them.

Biocompatible Materials↗