Biomedical subjects

N Philp

Publications and source records attributed to N Philp.

10 recordsLinked to original sources

PubMed1998

Integrins and development: how might these receptors regulate differentiation of the lens.

Integrins transduce both internal signals and signals from the matrix. These interactions between integrins, their extracellular matrix ligands, and their cytoskeletal partners play an important role in the regulation of cellular differentiation. We have shown them to be important in lens cell differentiation. In the lens capsule there is a compartmentalization of matrix components with fibronectin, primarily localized to the anterior capsule, and tenascin in the posterior capsule. Integrins are developmentally regulated in the lens. alpha 5 beta 1 integrin, like fibronectin, is primarily associated with the lens epithelial cells, where together they are likely to be important in regulation of adhesion and proliferation. alpha 6A beta 1, the integrin laminin receptor, is expressed at its highest levels in the equatorial epithelium and the peripheral fiber cells, both migratory populations. Because laminin is uniformly distributed in the lens capsule, such changes in alpha 6A integrin expression are likely critical to the cell's ability to regulate its response to laminin in the matrix. The organization of cytoskeletal molecules associated with the integrin cytoplasmic face also changes with development. In the epithelial regions of the lens, where the initiation of lens cell differentiation occurs, expression of the cytoskeletal proteins involved in cell-substrate interactions, talin, alpha-actinin, and the signaling proteins, are high. In the fiber cell region of the lens, where the cells establish stable cell-cell contacts, vinculin predominates and becomes highly associated with the cytoskeletal fraction. The role of integrins in lens development is not only regulated by changes in the expression of different integrin receptors but is also closely correlated with the expression and organization of the molecules with which they associate.

Animals↗

PubMed1997

Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of developing muscle cells.

3T3 Cells↗

PubMed1996

Left-right asymmetric localization of flectin in the extracellular matrix during heart looping.

The early embryo is initially bilaterally symmetrical. One of the first distinct indications of asymmetry in the embryo occurs during heart looping. The midline tubular heart begins to bend to the right to form a C-shaped structure around 30 hr of development in the avian model. A molecular basis for heart asymmetry and direction of looping is not known, although factors inherent to the myocardium are believed to underlie looping. A left-right asymmetric localization of a specific molecule in the bilateral heart forming regions has not been reported previously. One molecule that we are calling flectin (flectere, in L., to bend or to loop) shows a bilateral asymmetric localization early in the heart forming mesoderm and continues to be expressed asymmetrically in a highly organized manner in the cardiac jelly during heart looping. This large extracellular matrix molecule has been identified using a monoclonal antibody F-22 (Mieziewska et al., 1994a,b). Flectin shows a discrete spatiotemporal pattern of extracellular matrix expression during avian heart development. An asymmetric expression of flectin is observed during heart development at stage 7+/8- (approximately at 24 hr of development around the 3-somite stage). It is predominantly expressed in the left precardiac mesoderm at this developmental period. Between stages 12 and 14, flectin continues to be asymmetrically expressed in the myocardium and is localized at high levels on the basal side of the myocardium and within the cardiac jelly extending to the endocardial cell surfaces. In the same plane of the looping part of the heart it is differentially organized within the cardiac jelly on the convex side and in the outer loop areas. A reduced expression is apparent anteriorly and posteriorly along the tubular heart. The initial asymmetry of localization is maintained throughout the tubular heart. At stage 22 (Embryonic Day 3.5), intensity of immunolocalization of flectin is significantly decreased, with left-right asymmetry becoming less discernible or absent. It again is expressed in Day 10 embryonic hearts. Flectin expression appears to be modulated by retinoids. In vitamin A-deficient quail embryonic hearts that do not loop (Dersch and Zile, 1993; Twal et al., 1995), flectin protein expression is decreased and disorganized, as are other extracellular matrix components comprising the cardiac jelly.

Animals↗

PubMed1995

Developmental expression and molecular cloning of REMP, a novel retinal epithelial membrane protein.

The retinal pigment epithelium (RPE), like other transport epithelia, has a polarized distribution of membrane and cytoskeletal proteins. The establishment of a polarized phenotype is an essential step in the differentiation of the RPE and the development and maintenance of visual function. Using a monoclonal antibody (MAb 3C4) we have identified a novel membrane protein that is uniquely expressed in chick RPE. We have referred to this protein as REMP for retinal epithelial membrane protein. In these studies we characterized the expression and distribution of this protein during embryonic development and determined its primary structure by cDNA cloning. The developmental expression of REMP was examined by immunocytochemical localization. REMP was first detected in the chick RPE at Embryonic Day 5 (E5) in both apical and basolateral membranes. By E14 the distribution of REMP was restricted to the basolateral surface of the RPE cells. Biochemical fractionation and surface labeling of RPE cells suggested that REMP was an integral protein. The gene encoding REMP was isolated from an E15 chick RPE cDNA library, cloned into lambda gt11, and screened with MAb 3C4. The cDNA was sequenced and found to contain one 1350-bp open reading frame encoding for a 450-amino-acid protein. The deduced amino-acid sequence of REMP shares 32.9% identity with MCT1, a monocarboxylate transporter (Garcia, Goldstein, Pathak, Anderson, and Brown, Cell, 76, 865-873, 1994). By Northern blot analysis, REMP mRNA was detected only in RPE cells. There was an increase in the expression REMP transcript during development but when RPE cells were grown in primary culture the expression of REMP was turned off. The unique expression of REMP in the RPE in vivo would suggest a role for this protein in development and maintenance of normal retinal function.

Amino Acid Sequence↗

PubMed1994

Redistribution of insoluble interphotoreceptor matrix components during photoreceptor differentiation in the mouse retina.

The development of the nervous system is largely influenced by the extracellular matrix (ECM). In the neural retina, the photoreceptors are surrounded by a unique ECM, the interphotoreceptor matrix (IPM). The IPM plays a central and possibly crucial role in the development, maintenance and specific function of the photoreceptors. Therefore, the characterization of IPM components is necessary to understand the mechanisms regulating photoreceptor differentiation. The IPM in the mouse retina was examined during photoreceptor morphogenesis with the monoclonal antibody (MAb) F22, which recognizes a 250 kDa component of the interphotoreceptor matrix. The binding pattern of MAb F22 revealed a striking redistribution in the expression of the 250 kDa F22 antigen in late stage of postnatal photoreceptor differentiation in the mouse retina. The F22 staining was detectable in the IPM around the inner segments on the third postnatal day (P3). The MAb F22 initially labeled the region around inner segments, but as the outer segments elongated, the F22 distribution became concentrated to the matrix around the rod and cone outer segments until P16-17. At P17, the F22 label around rods began to disappear, while the label around cones became more defined. The shift in label distribution was largely completed by P20. Residual rod-associated label disappeared within a few days. In the adult animal, the F22 antibody labeled the cone-associated matrix only, and this labeling pattern remained stationary. The change in the distribution of MAb F22 demonstrated by immunolabeling was not accompanied by changes in the size of the molecule; F22 antigen isolated from the IPM of P13-15, and from adult IPM migrated with the same molecular weight on SDS gels. The distribution of MAb F22 was compared to that of chondroitin sulfate proteoglycans which are abundant in the IPM. The labeling patterns of MAbs CS-56, C6-S and C4-S were distinct from that of MAb F22. A general decrease of the label intensity was seen with two chondroitin sulfate MAbs (CS-56 and C4-S) between 16 days and 4 months, but a total loss of rod-associated label was not observed. All three chondroitin sulfate MAbs labeled the retina at embryonic day (E) 11.5-13.5, a time of outgrowth of ganglion cell axons, but the F22 antigen was not detected in the retina at this stage of development. The results demonstrate that the F22 and the chondroitin sulfate antibodies are recognizing different molecules that have distinct roles in retinal morphogenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Diethylene glycol distearate (DGD): a versatile embedding medium for retinal cytochemistry.

Embedment in diethylene glycol distearate (DGD) was shown to be highly desirable and versatile for retinal cytochemical studies, including in situ hybridization, immuno- and lectin cytochemistry. This method allows for preservation of fine tissue detail as well as good reaction sensitivity. It appears to be more suitable than most other methods currently used for light microscopic retinal cytochemistry.

Animals↗

PubMed1993

Evaluation of integrin molecules involved in substrate adhesion.

Integrins were cross-linked to their extracellular matrix ligands using non-penetrating chemical cross-linkers. This procedure did not disturb the distribution of integrin in the adhesion structure and adhesion plaque integrin staining remained even when the cultures were extracted with ionic detergents. 80-90% of the beta 1 integrin in the cross-linked culture was extracted with RIPA buffer and the remaining 10-20% was recovered following reversal of the cross-linking. This separated two distinct integrin pools, one which can be cross-linked to substrate bound extracellular matrix and one which is not. The specificity of this procedure for cross-linking of integrins involved in substrate adhesion was demonstrated using NIH 3T3 cells which express both alpha 5 beta 1 and alpha 6 beta 1 integrins. alpha 6 was cross-linked only in cells plated on laminin whereas alpha 5 was cross-linked when fibronectin was present. Using antisera directed to the cytoplasmic domains of either alpha 5 or beta 1 integrin, it was demonstrated that these domains can be blocked in the intact cell but the blocking can be removed using ionic detergent extraction after chemical cross-linking. The extracellular matrix associated with the substrate surface but not that associated with the media exposed surface is both cross-linked and retained on the plastic dish following cross-linking.

3T3 Cells↗

PubMed1992

G-actin pool and actin messenger RNA during development of the apical processes of the retinal pigment epithelial cells of the chick.

It has been suggested that during development an increase in the pool of G-actin may drive the elongation of actin-containing processes which occur in several types of epithelial cells. The apical processes of chick retinal pigment epithelial (RPE) cells elongate during the last 7 days of embryonic life (E15-E21) reaching lengths of 20 microns or more by hatching (E21). F-actin bundles form the cores of these processes. We followed the elongation by measuring F-actin in the cells and cytoskeletons. In correlation with this, we studied by DNAse assay the levels of monomeric actin in supernatants of cell extracts from E13, before elongation starts, to E17, when elongation is well underway. Total F-actin increased 1.9-fold over this time period and cytoskeletal actin increased 2.5-fold. In supernatants from extracts of E13 RPE the monomeric actin concentration was 51 +/- 0.5 micrograms/ml. From estimates of cell volume we calculated the cellular monomeric actin concentration at E13 as at least 510 micrograms/ml (13 microM). We compared this with monomeric actin levels in extracts from RPE at E15 and E17. Allowing for the estimated increase in cell volume, our data show little overall change in cellular monomeric actin concentration at these times. Changes in the level of actin mRNA were measured over the same time period. Normalized to equal RNA, we found a twofold increase in beta actin mRNA and a four- to fivefold increase in message for gamma actin at E17 as compared to E13. In summary, we show that (1) there is a substantial pool of monomeric actin in these epithelial cells before elongation starts; (2) process elongation is not associated with a significant change in the size of this pool; and (3) process elongation is associated with a significant increase in actin mRNA.

Actins↗

PubMed1988

S-antigen in a hereditary visual cell disease. Immunocytochemical and immunological studies.

S-antigen is a photoreceptor-specific and potentially autoantigenic protein. Using light microscopic immunocytochemistry, the localization of S-antigen was studied in the retinas of normal dogs and Irish setters affected with rod-cone dysplasia, a hereditary retinal degeneration characterized by abnormal cGMP metabolism and arrested outer segment differentiation. Normal and affected dogs were also tested for the presence of humoral and cellular immunity to S-antigen. S-antigen was present in both rods and cones during inner and outer segment differentiation, but there was an apparent loss of immunoreactivity in cones as the retina matured. The developmental appearance and localization of S-antigen in affected retinas was similar to that of normals. S-antigen immunoreactivity decreased during the early stages of rod loss (39-57 days), but was still present in photoreceptor somata in the late stages of retinal degeneration. No significant difference was found between normal and affected setters in humoral immunity to S-antigen, indicating that it probably does not stimulate autoimmunity in red 1. Because normal dog lymphocytes failed to respond when sensitized to bovine S-antigen, cellular immunity to S-antigen in this disease cannot be ruled out.

Animals↗

PubMed1980

Rupture of the urinary bladder in children. The importance of the double lesion.

We report 10 cases of rupture of the urinary bladder in children. Only one patient was a girl. The rupture was caused by a road accident in 6 cases and occurred "spontaneously" in 4. Rupture of the bladder may be difficult to diagnose. In many patients the vesical rupture is not the only abnormality in the urinary tract and a thorough investigation is necessary. The majority of the bladder ruptures in these children were intraperitoneal, which contrasts with the reported experience in adults.

Child↗