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Biomedical subjects

A S Menko

Publications and source records attributed to A S Menko.

At least 19 recordsLinked to original sources
PubMed2001

Loss of alpha3beta1 integrin function results in an altered differentiation program in the mouse submandibular gland.

Mammalian submandibular gland (SMG) development leads to the establishment of highly organized secretory acinar and nonsecretory ductal epithelial cells. The ability of maturing salivary epithelial cells to attain their differentiated state has been shown to depend, in part, on interactions between extracellular matrix (ECM) proteins and their integrin receptors. In a search for key regulators of salivary cell lineage, we have studied alpha3beta1 integrin, a receptor for the basement membrane protein laminin, by characterizing embryonic day 18 (E18) SMGs isolated from mice carrying a targeted mutation in the alpha3 integrin gene. Transmission electron microscopy studies showed that the mutant SMGs exhibited an aberrant differentiation phenotype with defects in the apical-basal polarity axis and in the basement membrane. Based on immunohistochemistry and Western blot analyses, the alpha3beta1-deficient SMGs had altered expression and/or localization of several ECM and adhesive molecules, including laminin beta1, fibronectin, alpha5 integrin, and E-cadherin. These changes correlated with alterations in the activation state of Ras-extracellular signal-regulated kinase (ERK), as well as the expression and/or localization of Cdc42 and RhoA, two Rho GTPases that regulate the organization of the actin cytoskeleton. We conclude that alpha3beta1 is required for normal salivary cell differentiation and that its absence affects multiple components of adhesive complexes and their associated signalling pathways.

Actins↗
PubMed2000

Decreases in endogenous opioid peptides in the rat medullo-coerulear pathway after chronic morphine treatment.

Several biochemical changes have been described in noradrenergic neurons of the locus coeruleus (LC) after chronic morphine treatment. Changes in neurochemical expression in opioid afferent projections to the LC may be equally important in modulating noradrenergic neurons during chronic opiate exposure. To test the hypothesis that opioid peptides in LC afferents are altered after chronic opiate administration, we exposed adult male rats to either morphine or placebo pellets for 5 d. Tissue sections through the LC were processed for peroxidase or gold-silver labeling of methionine(5)-enkephalin (met-ENK) and analyzed using light or electron microscopy, respectively. Light level densitometry and ultrastructural analysis showed that there was a significant decrease in immunolabeling for ENK in LC-afferent terminals of morphine-treated rats. Western immunoblot analysis confirmed that protein levels for both leucine(5)- and methionine(5)-ENK were significantly decreased in tissue samples containing the LC after chronic morphine treatment. To test whether decreases in ENK protein expression were mirrored by decreases in gene expression, Northern blot analysis of preproenkephalin (PPE) mRNA was conducted in tissue samples obtained through the medulla, a brainstem area that contains the major opioid afferents to the LC. PPE mRNA was reduced in samples obtained from morphine-treated rats. Finally, in situ hybridization experiments confirmed significant decreases in PPE mRNA expression in the nucleus paragigantocellularis, a region known to provide a robust opioid input to the LC. These data suggest that there is a decrease in the synthesis of the opioid peptide mRNA and protein in the medullo-coerulear pathway after chronic exposure to morphine. Such alterations in opioid peptide levels during opiate dependence may contribute to the observed hyperactivity of LC neurons during opiate withdrawal.

Animals↗
PubMed2000

N-cadherin function is required for differentiation-dependent cytoskeletal reorganization in lens cells in vitro.

Members of the cadherin family of cell adhesion molecules participate in calcium-dependent cell-cell adhesions that are necessary for the cell sorting events that regulate early developmental processes. Although individual cadherin molecules have been shown to participate in tissue histogenesis, the regulation of function of these receptors in cell differentiation has been more difficult to identify. We have determined that N-cadherin linkage to the cytoskeleton is correlated with lens cell differentiation in vivo. Through the use of a chick embryo lens culture system that mimics differentiation in vivo, we have determined that N-cadherin linkage to the cytoskeleton is altered and lens differentiation is blocked by function-blocking antibodies to N-cadherin. In the presence of the N-cadherin function-blocking antibody, NCD-2, both N-cadherin and filamentous actin are prevented from organizing at the cortical membranes. This correlates with an inhibition of lens morphogenesis and differentiation. These results are paralleled by changes in the expression of the molecular components of the cadherin-catenin complex and their linkage to the actin cytoskeleton. In the presence of NCD-2, expression of N-cadherin, alpha-catenin, and beta-catenin is inhibited and their association with the cytoskeleton blocked. Overall cadherin expression, however, remains unchanged as demonstrated by studies with a pan-cadherin antibody. This is accompanied by an increase in expression of the cadherin cytoskeletal protein plakoglobin. Although the cells have tried to compensate for the loss of N-cadherin by up-regulation of another cadherin(s) and plakoglobin, this is unable to compensate for N-cadherin function. The data strongly suggest that N-cadherin and its associated cytoskeleton play an important role in the differentiation process that leads to the formation of the crystalline lens.

Actinin↗
PubMed2000

Differential expression of N- and B-cadherin during lens development.

PURPOSE: To analyze the dynamics of N- and B-cadherin cell adhesion molecule expression and cytoskeletal interaction during embryonic chick lens development. METHODS: Localization of N- and B-cadherin, F-actin, and connexin 56 were determined by immunohistochemistry of developing lenses or immunocytochemistry of differentiating primary lens cultures. Biochemical analysis of cytoskeletal linkage of N- or B-cadherin was assessed by differential detergent extraction, electrophoresis, and immunoblotting. RESULTS: The results indicate that although both cadherins are expressed throughout lens development, N-cadherin expression detected was similar in both lens epithelial and fiber cells, whereas B-cadherin was preferentially localized to the lens fiber cells. During differentiation, both cadherins become increasingly associated with the lens cytoskeleton, as indicated biochemically by a transition from largely Triton X-100-soluble to Triton X-100-insoluble pools and immunocytologically by cadherin localization to cell-cell borders and colocalization with the actin cytoskeleton. Although a significant fraction of N-cadherin remains Triton X-100-soluble as the lens cells differentiate, B-cadherin becomes resistant to extraction by both Triton X-100 as well as RIPA buffers. As detected immunocytochemically in lens cell cultures, the temporal localization of N-cadherin to cell-cell interfaces precedes that of B-cadherin. Furthermore, temporal localization of B-cadherin, as opposed to N-cadherin, to cell-cell borders more closely parallels that of connexin 56 in vitro as well as in vivo. CONCLUSIONS; These results suggest that while both N- and B-cadherin are expressed during lens cell differentiation, both their patterns of expression as well as their cytoskeletal association differ between epithelial and fiber cells.

Actins↗
PubMed1999

alpha6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching.

The developing chicken embryo lens provides a unique model for examining the relationship between alpha6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that alpha6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell-cell interfaces of the differentiating cortical lens fiber cells. Both alpha6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of alpha6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens alpha6 integrin was uniquely localized along the cell-cell borders of the differentiating fiber cells, similar to beta1. alpha6beta4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of alpha6A and alpha6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that alpha6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with alpha6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that alpha6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of alpha6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the alpha6A isoform remained high until the cells became terminally differentiated. alpha6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of alpha6B relative to alpha6A provides a developmental switch in the process of lens fiber cell differentiation.

Animals↗
PubMed1999

Differential expression of proliferative, cytoskeletal, and adhesive proteins during postnatal development of the hamster submandibular gland.

Although the submandibular gland (SMG) plays important exocrine and endocrine roles, little is known about the molecular details underlying its development. Previously, we reported that in the postnatally developing hamster SMG, GPT, the protein product of the first N-glycosylation gene, ALG7, was an in vivo marker for salivary cell proliferation. Here we investigated the proliferative, cytoskeletal, and adhesive changes during SMG postnatal development. The cellular localization and abundance of GPT, filamentous actin, and beta1 integrin receptor were examined using confocal microscopy and immunoblotting. In neonatal glands, high GPT levels marked extensive cell proliferation throughout the tissue. The apical regions of immature salivary cells displayed intense actin staining, while most of the beta1 integrin was diffusely distributed throughout the tissue. As development proceeded, discrete regions of the gland expressed attenuated levels of GPT, an increased organization of actin to the cell cortex, and beta1 integrin to the basal lamina. In the adult SMG, differentiated salivary cells displayed low levels of GPT and actin. While the abundance of beta1 integrin remained unchanged throughout development, in the adult, it was found exclusively in regions where cells contact the basal lamina. These data indicate that SMG development entails regionalized cell proliferation and polarization, and that these processes are temporally and spatially coordinated with the establishment of stable cell-substratum interactions.

Actins↗
PubMed1997

Expression of schwannomin in lens and Schwann cells.

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder characterized by the development of bilateral vestibular schwannomas, meningiomas, ependymomas and juvenile lens opacities. The NF2 gene encodes a tumor suppressor protein, schwannomin (or merlin), with sequence homology to erythrocyte band 4.1, talin, ezrin, moesin and radixin. Using an antibody that recognizes the carboxy-terminal epitope of isoform 1 of schwannomin, we looked at its expression in lens and Schwann cells, two cell-types affected by the NF2 phenotype. Schwannomin was detected as an approximately 80 kDa protein in both cytoplasmic and cytoskeleton fractions. Indirect immunofluorescence localized schwannomin to the cytoplasm and was frequently observed in dynamic cellular regions such as leading edges and ruffling membranes. Its level of expression in the lens inversely correlates with the degree of lens cell differentiation suggesting a role for schwannomin in differentiation-specific events.

Animals↗
PubMed1995

Regulation of integrin alpha 5 beta 1 affinity during myogenic differentiation.

The antigen recognized by U1 alpha, a monoclonal antibody to the alpha chain of a chicken integrin fibronectin receptor, was identified as alpha 5. It identifies the same polypeptide as antisera raised to a sequence from the alpha 5 cytoplasmic domain. The U1 alpha antibody has the unusual functional property for alpha chain antibodies of enhancing the binding of alpha 5 beta 1 for its ligand fibronectin. U1 alpha was used to examine the function of alpha 5 beta 1 during myogenic differentiation. As myogenic cells differentiated from replicating myoblasts to bipolar myocytes there was a decrease in their adhesion to the substrate caused by inactivation of alpha 5 beta 1, which could be reversed by treatment of the cells with U1 alpha. The U1 alpha induced increased adhesion to fibronectin but did not inhibit the differentiation process as measured by formation of myotubes. However, U1 alpha did interfere with both cell migration and morphogenesis of myotubes. The resulting myotubes were smaller, more branched, and showed less regular alignment of nuclei. The results suggest that the ability of the cell to regulate alpha 5 beta 1 affinity is critical to myogenic morphogenesis.

Animals↗
PubMed1995

Beta 1 integrins in epithelial tissues: a unique distribution in the lens.

Integrins have been shown to play a role in directing and maintaining cell differentiation and polarization. The embryonic lens provides a good system in which to examine their role in epithelial cell differentiation, because all stages of lens development are represented in an individual embryonic lens. Therefore, we examined the expression and distribution of beta 1 integrin heterodimers in both the lens epithelium and the differentiated lens fiber cells. In lens epithelial cells beta 1 integrin was found to be localized to all membrane surfaces. Lens fiber cells contained beta 1 integrin all along their lateral borders as well as at the site of their attachment to the lens capsule and at their interface with lens epithelial cells. The distribution of beta 1 integrin in the lens was distinct from that observed in simple epithelia, the retinal pigment epithelium (RPE), kidney, and intestine, where it was limited to a basal lateral localization. We examined the specific beta 1 integrin heterodimers expressed in the lens by Western blot analysis for the integrin alpha subunits following beta 1 immunoprecipitation and compared them with those expressed in RPE cells. In the lens we detected alpha 3 and alpha 6 subunits but not alpha 1, alpha 5, or alpha v. When the lens was separated into epithelial and fiber cells, we found that alpha 3 was expressed at a higher level in the epithelial cells, while alpha 6 was primarily associated with the fiber cells. In the RPE the primary beta 1 integrin detected was alpha 3. Unlike in lens and kidney, alpha 6 beta 1 integrin in RPE cells was expressed only at a low level. alpha v was also expressed in RPE cells but not as a beta 1 heterodimer. As in the lens, neither alpha 5 beta 1 nor alpha 1 beta 1 integrin was detected in RPE. Both lens and RPE cells express a specific subset of beta 1 integrin heterodimers which are likely to be important to the initiation and maintenance of their differentiated phenotype.

Animals↗
PubMed1993

Alpha 5 integrin is a critical component of adhesion plaques in myogenesis.

We investigated the distribution and expression of alpha 5 beta 1 and alpha 3 beta 1 integrin in differentiating myogenic cells in culture. The myogenic cells expressed both alpha 5 and alpha 3 integrin subunits with the same molecular sizes as those expressed by chicken embryo fibroblasts (CEF). However, the ratio of total alpha 5 to alpha 3 was threefold higher in the muscle cultures than that in CEF cultures. A new method is described whereby adhesion plaque-associated integrin was cross-linked to its extracellular matrix ligand on the substrate using a nonpenetrating cross-linker, BS3, and integrin not involved in substrate adhesion as well as cytoskeletal proteins were removed with a zwitterionic detergent. alpha 5 and beta 1 integrin, but not alpha 3 could be cross-linked to fibronectin at adhesion plaques throughout myogenesis in culture. Alpha 5 beta 1 integrin was found only at the edge of myoblasts 4 hr after plating but became distributed under their entire surface by 1 day in culture. When the muscle cells became elongated, a morphology they express after the initiation of terminal differentiation, and as they began to fuse, alpha 5 was found redistributed in small adhesion plaques along the lateral edges of the postmitotic myocytes and early myotubes. In mature myotubes, which are large multinucleated branched structures, alpha 5 beta 1 integrin was localized to parallel streaks underneath their entire substrate surface. Throughout the different stages of myogenesis, vinculin colocalized with alpha 5 beta 1 integrin in adhesion plaques, but alpha-actinin only colocalized to the adhesion plaques in myoblasts, not in myotubes. These studies suggest that alpha 5 beta 1 integrin through its dynamic interaction with both fibronectin and the cytoskeleton is important for both the signals which initiate the differentiation process and for subsequent morphological and structural changes during the differentiation process.

Animals↗
PubMed1993

Beta 1 integrins mediate chondrocyte interaction with type I collagen, type II collagen, and fibronectin.

Chondrocytes isolated from the cephalic region of sterna from 14-day-old chick embryos used beta 1 integrins and required either Mg2+ or Mn2+ for attachment to plates coated with type I collagen, type II collagen, and fibronectin. beta 1 integrin was concentrated in adhesion plaques of the chondrocytes plated on type I collagen, type II collagen, and fibronectin substrates. Chondrocytes expressed at least 3 alpha-subunits, including alpha 3, alpha 5, and putative alpha 2. alpha 5, but not alpha 3, had a higher molecular weight in chondrocytes than in fibroblasts. Levels of alpha 3 and alpha 5 were about 25-30% of that in fibroblasts. When the chondrocytes were cultured in the presence of ascorbate in suspension, the cells aggregated into clusters. This aggregation was dependent on beta 1 integrin and type II collagen.

Amino Acid Sequence↗
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↗
PubMed1988

Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src.

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.

Animals↗
PubMed1987

Occupation of the extracellular matrix receptor, integrin, is a control point for myogenic differentiation.

Replicating chicken embryo myoblasts were treated with a monoclonal antibody, CSAT, which recognizes and blocks the function of the beta subunit of integrin. In the presence of this antibody, myoblasts continued to replicate and did not fuse or produce muscle-specific meromyosin. This block to normal differentiation was readily reversed by the removal of the antibody. The reversed cells exhibited increases in desmin and meromyosin, and fused to produce contracting myotubes. The data indicate that the interaction of integrin, present on the membrane of replicating myoblasts, with extracellular matrix on the substrate is essential to initiate the terminal stages of myogenic differentiation.

Animals↗
PubMed1987

Junctions between lens cells in differentiating cultures: structure, formation, intercellular permeability, and junctional protein expression.

We previously described cultures of chick embryo lens cells which displayed a marked degree of differentiation. In this report, the junctions found between the lens fiber-like cells in the differentiated "lentoids" are characterized in several ways. Thin-section methods with electron microscopy first demonstrated that numerous, large junctions between lentoid cells accompanied the other differentiated features of these cells. Freeze-fracture techniques, including quantitative analysis, then revealed that (a) junctional particles were loosely arranged as is typical of fiber cells, (b) the population of individual junctional areas in culture was indistinguishable from that found in 10- to 12-day chick embryo lenses, and (c) apparent junction formation occurred during the development of the lens cells, with lacy arrays of particles being associated with fiber-like junctions. In addition, gap junctions with hexagonally packed particles, typical of lens epithelial cells, largely disappeared during the course of differentiation. Injection of tracer dyes into lentoid cells resulted in rapid intercellular movement of dye, consistent with functional cell-to-cell channels connecting lentoid cells. During the development of the lens cells in culture, as junction formation occurred, an increase of approximately eight-fold in MP28 protein was observed within the cells. These combined results indicate that (a) extensive lens fiber junctions and functional cell-to-cell channels are found between differentiated lentoid lentoid cells in vitro, (b) lens fiber junctions appear to form during the course of lens cell differentiation in culture, (c) a significant increase occurs in the putative junctional protein before the cultures are highly developed, (d) the increased levels of MP28 and junction formation may be required for the full expression of the differentiated state in the lens fiber cell, and (e) this culture system should prove to be valuable for additional experiments on lens junctions and for other studies requiring the development of lens fiber cells in vitro.

Animals↗
PubMed1985

Junctions between lens fiber cells are labeled with a monoclonal antibody shown to be specific for MP26.

A monoclonal antibody (mcAb) that recognizes an intracellular domain of the major lens membrane protein in both chicken and bovine lenses is described. Mice were immunized with chicken lens fiber cell membranes that had been washed with 7 M urea. Hybridomas were screened by means of enzyme-linked immunosorbent assays and the molecular specificities of the mcAbs were determined using electrophoretic transfer procedures, "Westerns." One of these mcAbs, an IgG designated B2, reacted with a single band of 28,000 Mr from the chicken embryo lens (MP28) and the analogous 26,000 Mr protein in the bovine lens (MP26). Monoclonal B2 was shown to be specific for these proteins, since (a) heating in SDS caused MP26 to aggregate and reduced B2 binding to the protein band at an Mr of 26,000 in Western transfer analysis; (b) apparent dimers were bound by B2 in Western transfers; (c) soluble protein fractions from the lens contained no detectable B2 antigens; and (d) a cyanogen bromide fragment of MP26 was bound by B2. Studies with several proteases indicated that the antigenic site for B2 resides on a 2-kd, protease-sensitive region at the C-terminal end of MP26 and MP28. Evidence for B2 binding on the cytoplasmic side of the membrane comes from labeling studies done at the ultrastructural level. These studies, utilizing indirect methods with peroxidase and colloidal gold markers, clearly demonstrated that B2 labels two types of junctional profiles. In our calf lens membrane preparations after tannic acid staining, the predominant type (80%) measured 16-18 nn thick, with the second type measuring only 12-14 nm. Chick embryo lens cells that had differentiated in vitro and formed groups of lens fiber-like cells (termed lentoids), fluoresced brightly only when they had been permeabilized before labeling with B2 and a fluorochrome-conjugated antibody. This binding was concentrated at the plasma membranes of cells within the lentoids, even outside areas of cell-cell contact. Surrounding epithelioid cells were not stained. Solubilized lens cultures, examined by Westerns, displayed a single immunoreactive band, which co-migrated with MP28.

Animals↗