Biomedical subjects

K T Bush

Publications and source records attributed to K T Bush.

30 records · Page 2Linked to original sources

PubMed1994

Epithelial inositol 1,4,5-trisphosphate receptors. Multiplicity of localization, solubility, and isoforms.

In an earlier subcellular fractionation study of epithelial tissue (liver and pancreas), we demonstrated that the inositol 1,4,5-trisphosphate receptor (IP3R) is found in association with biochemically distinct cellular membranes, including the endoplasmic reticulum (ER) and plasma membrane (Sharp, A. H., Snyder, S. H., and Nigam, S. K. (1992) J. Biol. Chem. 267, 7444-7449). To further characterize epithelial IP3Rs, we have now employed cultured Madin-Darby canine kidney (MDCK) cells, a well studied tight polarized epithelial cell type. Indirect immunofluorescence with an antiserum which specifically recognizes IP3R in MDCK cells by immunoblotting and immunoprecipitation gave an ER-like staining pattern as well as a basolateral plasma membrane-like staining pattern, the latter being particularly evident in highly confluent monolayers. In sections of adult rat kidney tubules a similar staining pattern was observed. Interestingly, whereas known basolateral proteins (Na+,K(+)-ATPase and the facilitated glucose transporter) gave a continuous basolateral staining pattern, that seen for IP3R was discontinuous (punctate). A highly similar staining pattern was observed for the caveolar protein, caveolin, suggesting that the punctate basolateral plasma membrane-like staining pattern observed for IP3R reflects its localization to basolateral caveolae. Biotinylation of non-permeabilized and permeabilized MDCK cells, followed by immunoprecipitation of IP3R and detection with streptavidin, indicated that while most IP3R is localized to biotin-inaccessible compartments (i.e. ER), a fraction (10-20%) of IP3R was accessible to externally added biotin primarily from the basolateral side. This result is compatible with the dual ER and basolateral caveolar localization suggested by immunocytochemistry, although it does not exclude the presence of some IP3R in the basolateral plasma membrane as well. Solubility studies revealed IP3R to be considerably more insoluble than the basolateral proteins, Na+,K(+)-ATPase and the liver cell adhesion molecule, as well as the cytoskeletal proteins, ankyrin and fodrin. In the most insoluble fraction, IP3R was found along with caveolin, further supporting the notion that part of the cellular IP3R pool associates with caveolae. Since multiple localizations of IP3R within a cell might reflect the existence of multiple isoforms, polymerase chain reaction amplification of first strand cDNA with primers specific for the three isotypes of IP3R was performed. All three isoforms of IP3R were expressed in the homogeneous population of MDCK cells. The existence of distinct membrane localizations and multiple isoforms of IP3R within the same cell type suggests an explanation for the complex spatiotemporal patterns of Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ pools in epithelial and other cells.

Animals↗

PubMed1994

A set of endoplasmic reticulum proteins possessing properties of molecular chaperones includes Ca(2+)-binding proteins and members of the thioredoxin superfamily.

The major proteins in the lumen of the endoplasmic reticulum (ER) are thought to function in Ca2+ sequestration or as "molecular chaperones" in the folding and assembly of membrane or secreted proteins. Based on the ability of many chaperones to bind selectively to unfolded proteins and to dissociate from them upon ATP hydrolysis, we developed an affinity chromatography method to isolate proteins with these characteristics from pancreatic or liver ER. Seven ER proteins bound selectively to denatured protein columns and were specifically eluted by ATP (10(-6) M) but not by a nonhydrolyzable ATP analog. These proteins were identified with antibodies and microsequencing as the ER chaperone BiP (grp78), grp94, calreticulin, a novel 46-kDa protein that binds azido-ATP, as well as three members of the thioredoxin superfamily: protein-disulfide isomerase, ERp72, and a previously reported 50-kDa protein (p50). This set of seven proteins bound to and was eluted with ATP from a variety of denatured proteins, including histone, gelatin, alpha fetoprotein, thyroglobulin, lysozyme, casein, and IgG. The release of grp94, protein-disulfide isomerase, ERp72, calreticulin, and p50 was stimulated by Ca2+ in the presence of ATP. These proteins thus appear to function as Ca(2+)-dependent chaperones, which may account for the Ca2+ and ATP requirement for protein folding in the ER.

Adenosine Triphosphate↗

PubMed1993

Localization of the FK506-binding protein, FKBP 13, to the lumen of the endoplasmic reticulum.

The function of the immunophilins, FKBP 12 and FKBP 13, which are binding proteins for the immunosuppressant drug FK506 and rapamycin, remains poorly defined, although it has been suggested that immunophilins and immunophilin-like proteins may play a role in protein sorting/folding and intracellular calcium ion regulation. As a first step towards understanding the function of FKBP 13, we studied its subcellular localization by immunoblotting of well-defined subcellular fractions from a canine pancreatic homogenate and immunocytochemical analysis of an overexpressed cloned cDNA for FKBP 13. Whereas FKBP 12 fractionated entirely into the cytosol, virtually all FKBP 13 was found in the rough microsomal fraction which consisted of highly purified rough endoplasmic reticulum (ER), along with several well-characterized ER markers [the immunoglobulin heavy-chain binding protein (BiP), grp 94 and ribophorin I]. Moreover, FKBP 13 co-banded with the ER markers on isopycnic sucrose gradients. By immunofluorescence, the overexpressed cDNA for FKBP 13 in Hela cells gave an ER-staining pattern highly similar to that of known ER proteins. Addition of the ligand FK506 did not appear to alter the distribution of FKBP 13. Separation of the ER luminal contents and membrane revealed FKBP 13 to be a luminal ER protein. Since the lumen of the ER is where the folding of membrane and secreted proteins occurs, as well as a major site of intracellular calcium storage, it seems possible that FKBP 13 may be involved in one of these functions.

Amino Acid Sequence↗

PubMed1992

Lipid droplets of neuroepithelial cells are a major calcium storage site during neural tube formation in chick and mouse embryos.

In situ precipitation of calcium (Ca2+) with fluoride and antimonate shows that Ca(2+)-specific precipitate is localized almost exclusively within lipid droplets of neuroepithelial cells during neural tube formation in chick and mouse embryos. The density of Ca2+ precipitate within lipid droplets is generally greater in the apical ends of cells situated in regions of the neuroepithelium that are actively engaged in bending. These findings suggest that lipid droplets, in addition to providing a source of metabolic fuel for developing neuroepithelial cells, also serve as Ca(2+)-storage and -releasing sites during neurulation.

Animals↗

PubMed1992

Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in rats.

The effect of the combination of total parenteral nutrition (TPN) and systemic sepsis on mucosal morphology and protein synthesis was investigated. Rats were given a standard TPN mixture consisting of glucose (216 kcal.kg-1.day-1), lipid (24 kcal.kg-1.day-1), and amino acids (1.5 g N.kg-1.day-1) for 5 days. On the 5th day the rats (n = 37) were randomized into four groups according to diet as follows: 1) control nonseptic on standard TPN, 2) control nonseptic on TPN with glutamine, 3) septic on standard TPN, and 4) septic with the TPN supplemented with glutamine. Twenty hours after the injection of Escherichia coli, the rats were given a 4-h constant infusion of [U-14C]leucine to determine the mucosal fractional protein synthesis rates. The following results were obtained. 1) Histological examination showed that systemic sepsis caused tissue damage to the ileum and jejunum. 2) Glutamine supplementation attenuated these changes. 3) There were no visible changes to the colon either from glutamine supplementation or sepsis. 4) Sepsis was associated with an increase in mucosal protein synthesis and decreased muscle synthesis. 5) Addition of glutamine to the TPN mix further increased protein synthesis in the intestinal mucosa of septic rats.

Animals↗

PubMed1991

A morphometric and computer-assisted three-dimensional reconstruction study of neural tube formation in chick embryos.

The origin of the driving forces for neural tube formation remains uncertain but is currently thought to involve the participation of microfilament bundles situated in the apical ends of neuroepithelial cells. In the work presented here, we show how morphometric measurements that map local variations in the apical geometry of neuroepithelial cells (especially apical constriction) can provide information on the distribution of motive forces within the neuroepithelium during neural tube formation. When used in combination with computer-assisted, three-dimensional reconstruction, it becomes possible to analyze the morphometric data from a dynamic, three-dimensional perspective. As an example application of this method, we have used morphometry to evaluate the effects of ionomycin on the developing neuroepithelium. Treatment of early (stages 6-8) chick embryos with 5 microM ionomycin was found to cause rapid bending of the neuroepithelium within 1 min of exposure and a dramatic acceleration of the normal sequence of neural tube formation. Electron microscopy and morphometry revealed that this acceleration was coincident with a marked increase in the local degree of apical constriction of neuroepithelial cells, presumably a consequence of enhanced contractile activity of apical microfilament bundles. This work shows that transient elevation of free calcium levels can accelerate the usual sequential phases of NT formation. The rapidity of the response (hours of normal development reduced to minutes), increased prominence of apical microfilament bundles, and the enhanced degree of apical constriction strongly support a direct causal role for apical microfilament bundles and apical constriction of neuroepithelial cells in bending of the neuroepithelium.

Actin Cytoskeleton↗

PubMed1990

Neural tube formation in the mouse: a morphometric and computerized three-dimensional reconstruction study of the relationship between apical constriction of neuroepithelial cells and the shape of the neuroepithelium.

Morphometry and computerized three-dimensional reconstruction were used to study the relationship between apical constriction of neuroepithelial cells and the pattern of bending of the neuroepithelium in the developing neural tube of the 12-somite mouse embryo. The neuroepithelium of the mouse exhibits prominent regional variations in size and shape along the embryo axis. The complex shape of most of the cephalic neural tube (e.g., forebrain and midbrain) is due to the coexistence of concave and convex bending sites whereas more caudal regions (e.g., hindbrain and spinal cord) generally lack sites of convex bending and have a relatively simple shape. The apical morphology of neuroepithelial cells was found to be correlated more closely with the local status of bending of the neuroepithelium than with the specific region of the neural tube in which they are located. In areas of enhanced apical constriction, microfilament bundles were particularly prominent. Morphometry revealed that patterns of bending of the neuroepithelium were correlated almost exactly with those of apical constriction throughout the forming neural tube. These findings support the idea that apical constriction of neuroepithelial cells, resulting from tension generated by microfilament bundles, plays a major role in bending of the neuroepithelium during neural tube formation in the mouse.

Actin Cytoskeleton↗

PubMed1989

Intrinsic and extrinsic factors collaborate to generate driving forces for neural tube formation in the chick: a study using morphometry and computerized three-dimensional reconstruction.

The formation of the neural tube, the rudiment of the entire central nervous system, is one of the earliest morphogenetic movements. The origin of the driving forces for this process remains uncertain, but recent studies suggest the involvement of both intrinsic and extrinsic factors. In the present study, we have used morphometry, analysis of stereopair photographs of whole embryos, and computerized three-dimensional reconstruction to investigate the factors which constitute the bulk of the driving forces for neural tube formation in the developing midbrain of Hamburger and Hamilton stages 5-9 chick embryos. Results support the notion that neural tube formation is driven by a coordinated interplay of intrinsic and extrinsic forces. Initial bending of the neural plate along the midline of the embryo and uplifting of the neural folds is accomplished primarily through the combined action of intrinsic forces (resulting from apical constriction of neuroepithelial cells) and extrinsic forces (mostly a passive consequence of head-fold formation). However, once in the uplifted position, curling over of neural folds and closure of the neural tube is driven largely by apical constriction-mediated (intrinsic) forces that are generated by cells in the midlateral walls of the forming neural tube.

Animals↗

PubMed1989

Biomechanical basis of diazepam-induced neural tube defects in early chick embryos: a morphometric study.

The biomechanical basis of diazepam (Valium/Roche)-induced neural tube defects in the chick was investigated using a combination of electron microscopy and morphometry. Embryos at stage 8 (four-somite stage) of development were explanted and grown for 6 hr in nutrient medium containing 400 micrograms/ml diazepam. Nearly 80% of these embryos exhibited neural tube defects that were most pronounced in the forming midbrain region and typified by a "relaxation" or "collapse" of neural folds. The hindbrain and spinal cord regions were less affected. Electron microscopy revealed that neuroepithelial cells in diazepam-treated embryos had smoother apical surfaces and broader apical widths than did controls. Morphometric measurements supported this observation and further showed that these effects were focused at sites within the wall of the forming neural tube that typically exhibit the greatest degree of bending and apical constriction (i.e., the floor and midlateral walls). Overall results indicate that neural tube defects associated with exposure to diazepam are due largely to a general inhibition of the contractile activity of apical microfilament bundles in neuroepithelial cells. These findings 1) emphasize the important contribution of microfilament-mediated apical constriction of neuroepithelial cells in providing the driving forces for bending of the neuroepithelium during neural tube formation and 2) suggest that agents or conditions that impair their contractile activity could play a role in the pathogenesis of certain types of neural tube defects.

Animals↗

PubMed1988

Immunoelectron microscopic localization of actin in neurites of cultured embryonic chick dorsal root ganglia: actin is a component of granular, microtubule-associated crossbridges.

Axons have been shown to contain substantial quantities of actin distributed along their length. However, the general lack of information on the structure and organizational state of this protein in axons has made it difficult to assign it a functional role. In the present study, we used electron microscopic immunocytochemistry (immunogold labeling) on neurites from cultured embryonic chick dorsal root ganglia to: (1) determine the distribution of actin in neurites: (2) identify actin-containing structures; and (3) reveal whether or not actin is associated preferentially with microtubules. Results show that actin is present throughout neurites but is organized primarily into short filaments that are localized almost exclusively to granular, microtubule-associated crossbridges. We propose that these short actin filaments are part of the framework of a supramolecular 'carrier complex' for the slow component b polypeptides. In addition, actin-containing crossbridges are often closely associated with the surfaces of membrane-bound organelles. This suggests that actin and microtubule-associated crossbridges are involved somehow in fast axonal transport, although the nature of their participation in this process still needs to be resolved.

Actins↗

PubMed1988

A morphometric study of cytoskeletal reorganization in rat sciatic nerve axons following beta,beta'-iminodipropionitrile (IDPN) treatment.

Electron microscopy and morphometry were used to study changes in the organization of cytoskeletal elements (microtubules (MTs) and neurofilaments (NFs] in myelinated rat sciatic nerve axons resulting from exposure to beta,beta'-iminodipropionitrile (IDPN). Adult male rats were injected subepineurially with IDPN and sacrificed at intervals. Electron micrographs of thin sections through injected nerve segments revealed that MTs segregated from NFs in axons and formed prominent clusters. Morphometric measurements of intermicrotubular distances within these clusters showed that adjacent MTs had a fairly uniform spatial separation that appeared to be maintained by granular MT-associated cross-bridges. In addition, MT clusters underwent a progressive, but reversible, compaction during the first 12 h of IDPN treatment.

Animals↗

PubMed1988

Time-lapse photographic study of neural tube closure defects caused by xylocaine in the chick.

Sequential changes in the morphology of early chick embryos were, for the first time, photographically recorded. Embryos were explanted at stage 8 (four-somite) or 9- (six-somite) of development using New's technique and grown in nutrient medium (thin albumen) with or without a teratologic dose (200 micrograms/ml) of xylocaine. They were photographed using a Nikon Diaphot inverted microscope equipped with both phase-contrast optics and photomicrographic accessories maintained in an incubator. It was found, among other things, that a characteristic neural tube closure defect often seen in the midbrain and anterior portion of the hindbrain of xylocaine (200 micrograms/ml)-treated chick embryos was a consequence of failure of the neural tube to withstand the tension generated by the rapidly expanding cephalic region, which occurred, regardless of the stage at explanation, when corresponding control embryos had advanced to stage 10+ (11-somite) of development.

Abnormalities, Drug-Induced↗