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W Ness

Publications and source records attributed to W Ness.

10 recordsLinked to original sources
PubMed1998

Essential mitotic functions of DNA topoisomerase IIalpha are not adopted by topoisomerase IIbeta in human H69 cells.

Unique functions of mammalian DNA-topoisomerases IIalpha and -beta are suggested by their distinct cellular distribution and chromatin binding at mitosis. Here, we studied H69-VP cells that, due to a homozygous mutation, express topoisomerase IIalpha mostly outside the nucleus. In these cells topoisomerase IIbeta showed a normal nuclear localization. However, at mitosis it diffused away from the chromatin despite the nuclear lack of the alpha-isoform. 80% of these cells performed chromosome condensation and disjunction with the aid of cytosolic topoisomerase IIalpha, which bound to the mitotic chromatin with low affinity. However, the genotype of these cells was highly polyploid indicating an increased rate of non-disjunction. In 20% of the mutant cells neither topoisomerase II isoform was bound to the mitotic chromatin, which appeared as an unstructured DNA spheroid unable to undergo disjunction and cytokinesis. Parental H69 cells expressing topoisomerase IIalpha inside the nucleus exhibited high affinity binding of the enzyme to the mitotic chromatin. Their genotype was mostly diploid and stable. We conclude (i) that high affinity chromatin binding of topoisomerase IIalpha is essential for chromosome condensation/disjunction and (ii) that topoisomerase IIbeta does not adopt these functions.

Antigens, Neoplasm↗
PubMed1998

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats.

Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na+,K+-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA+,K+-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting.

Animals↗
PubMed1998

Ischemia-induced phosphorylation and translocation of stress protein alpha B-crystallin to Z lines of myocardium.

It is becoming clear that stress proteins play a role in various aspects of postischemic myocardial recovery and that the cytoskeleton of cardiac myocytes is an important determinant for cellular survival during ischemia and energy depletion. In the present study, we addressed the question of whether the cytoskeleton-binding stress protein alpha B-crystallin may be involved in early cellular responses of rat and porcine myocardium to ischemia. Immunostaining and subcellular fractionation revealed a rapid ischemia-induced redistribution of alpha B-crystallin from a cytosolic pool to intercalated disks and Z lines of the myofibrils. This striking translocation of alpha B-crystallin from the cytosol to sites of the myofibrillar system that are known to be sensitive to ischemia-reperfusion injury was accompanied by a rapid shift of a fraction of alpha B-crystallin to a more acidic isoelectric point. This shift is caused by alpha B-crystallin phosphorylation, as identified by its augmentation in the presence of phosphatase inhibitors (vanadate, fluoride) and comigration of the acidic alpha B-crystallin form with the phosphorylated B1 form of lenticular alpha B-crystallin. In view of the chaperone-like function of alpha B-crystallin in conjunction with its high level of constitutive expression in the myocardium (1-2% of soluble protein content), we consider alpha B-crystallin an excellent candidate to play a role in early aspects of the protection of the myocardial contractile apparatus against ischemia-reperfusion injury.

Animals↗
PubMed1997

Sorting of actin isoforms in chicken auditory hair cells.

Most nonmuscle cells of higher vertebrates contain two different actin isoforms, beta- and gamma-cytoplasmic actin. The beta-isoform is with few exceptions the predominant isoform in nonmuscle cells and tissues. Perturbation of the beta:gamma ratio has been shown to affect the organization of bundled actin filaments indicating that the beta- and gamma-genes encode functionally distinct cytoarchitectural information. In the present study we localized by immunostaining beta- and gamma-actin in chicken auditory hair cells. These highly specialized cells serve as model system for studying certain developmental and structural aspects of a complex actin filament system with high architectural precision. We show that gamma-actin is the predominant actin isoform in auditory hair cells with an apparent beta:gamma ratio of approximately 1:2. gamma-Actin is not sorted and occurs in all three actin assemblies of the hair border, i.e. the cores of sensory hairs (stereocilia), the subjacent gel-like actin filament meshwork (cuticular plate) and the zonula adherens ring. In contrast to gamma-actin, the beta-isoform is specifically sorted to the actin filament core bundle of stereocilia that is extensively crosslinked by fimbrin. In view of recent studies showing that L-plastin, the leukocyte homolog of fimbrin, has a higher binding affinity for beta-actin than for gamma-actin, a mechanism is proposed for how hair cells might restrict formation of actin filament bundles to a single cellular site (i.e. the stereocilia). The limited level of expression of beta-actin in hair cells may help to prevent ectopic bundle formation in other cellular compartments.

Actins↗
PubMed1996

Dystrophin and the dystrophin-associated glycoprotein, beta-dystroglycan, co-localize in photoreceptor synaptic complexes of the human retina.

Mutations in the gene encoding for dystrophin, a membrane-associated cytoskeletal protein of muscle and several non-muscle cells, are the cause of Duchenne muscular dystrophy and Becker muscular dystrophy. Patients suffering from Duchenne muscular dystrophy have recently been shown to display an abnormal b-wave of the electroretinogram, suggesting that dystrophin is important for normal retinal transmission. In the retina, dystrophin has been localized in the outer plexiform layer where dystrophin co-localizes with postsynaptic markers of photoreceptor synaptic complexes. In the present study we addressed the question of whether two major dystrophin-associated integral membrane proteins of the muscular plasma membrane, beta-dystroglycan and adhalin, are also present in photoreceptor synaptic complexes. By double immunostaining and immunoblotting we show here that beta-dystroglycan is expressed in the human retina where it co-localizes with dystrophin in photoreceptor synaptic complexes most likely on the postsynaptic side. Adhalin was not detected in the retina. Since beta-dystroglycan is a member of a transmembrane supramolecular complex thought to be important for differentiation of the neuromuscular junction, it is an attractive hypothesis that dystroglycan (linked to dystrophin) might also play a similar role in differentiation of the photoreceptor synapse. A further outcome of this study is that beta-dystroglycan is not only present in the neuromuscular junction but also associated with a well-defined synaptic complex of the central nervous system. These findings indicate a more general role of this dystrophin-associated membrane protein in synaptic functions.

Adult↗
PubMed1994

Localization of protein kinase C in primary cultures of human keratinocytes in relation to cell contact proteins.

To investigate the role of protein kinase C (PKC), one of the key factors in cellular signalling, in the integrity of cell contacts, we studied the effects of PKC activation and inhibition on cell-cell and cell-substratum contacts. We localized PKC, actin and two major elements of zonula adherens (vinculin, E-cadherin) and focal contacts (vinculin) in primary cultures of normal human keratinocytes (nHEK) by fluorescent analogue cytochemistry. The activity of PKC was influenced by administration of TPA (12-O-tetradecanoylphorbol-13-acetate, a specific PKC activator) and H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine, a PKC inhibitor] for various periods of time. Our results show varying effects of TPA and H-7 on zonula adherens and focal contacts, suggesting differences in modulation of both types of adherens junctions by mechanisms partially involving PKC. In addition, TPA treatment of nHEK leads to changes in actin cytoskeletal organization.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Localization of adducin in epidermis.

On the cytoplasmic side of the plasma membrane of erythrocytes there is a dense protein filament matrix that maintains the shape of the cells. The main constituents of this system, actin and spectrin, which have also been detected in keratinocytes and fibroblasts, are known to be linked in erythrocytes in a network structure by additional proteins such as band 4.1 and adducin. The interaction between actin and spectrin, mediated by adducin, is regulated by calmodulin and protein kinase C. Because we have previously found adducin in cultured keratinocytes, we investigated epidermis by immunochemical techniques. We found adducin to be localized at cell-cell contact sites in epidermis using affinity-purified antibodies against human erythrocyte adducin. Immunofluorescence of epidermis revealed an intense fluorescence in the basal layer, whereas stratum spinosum and stratum granulosum showed moderate staining. There was intense staining at sites of cell-cell contact in cultured human keratinocytes. Immunoblot analysis indicated the presence of adducin polypeptides of 103 kd and 97 kd in epidermis, but in cultured keratinocytes only the higher molecular weight form could be detected. This study indicates adducin, a regulatory protein in erythrocytes, is also present in epidermis. Its localization suggests that it may be involved in the formation of the microfilament matrix of the membrane skeleton at cell-cell contact sites.

Actins↗
PubMed1993

Talin: adherens junction protein is localized at the epidermal-dermal interface in skin.

The interaction between cells of the epidermis and the basal lamina is important for the integrity of the skin. Several hereditary and acquired diseases show changes at the dermal-epidermal interface due to loss of adhesion between basal cells and the basement membrane. The structures mediating this interaction are hemidesmosomes, which have been extensively characterized by biochemical, molecular biologic, and morphologic techniques. Recently, however, a group of adhesion molecules that are distinct from hemidesmosomes and that mediate cell-matrix interactions was described in cultured fibroblasts, keratinocytes, and skin. These adhesion molecules, beta 1 integrins, have been shown to be present in the focal adhesion, a cell-matrix contact associated with microfilaments rather than intermediate filaments characteristic of hemidesmosomes. In cultured cells, integrins of the beta 1 family have been shown to be linked by a protein complex to actin filaments. In this study we describe the localization of talin, the binding protein for beta 1 integrins, and vinculin at the dermal-epidermal interface in skin with immunofluorescence and immunoblotting techniques. These data suggest the presence of a link between the cytoplasmic actin filament system in basal keratinocytes and the extracellular matrix.

Desmosomes↗
PubMed1993

Adherens junctions: demonstration in human epidermis.

Adherens junctions are intercellular and cell-matrix junctions that, like desmosomes and hemidesmosomes, mediate adhesion of cells to each other or to matrix structures. These junctions have been detected recently in cultured human keratinocytes, indicating that they may be of importance in epidermis. To investigate the localization of adherens junctions in normal epidermis, we examined human epidermis, human oral mucosa, and monkey esophagus for the presence of vinculin, a major protein of the intracellular plaques of adherens junctions that is thought to be present in all adherens junctions. Western blot analysis demonstrated vinculin in extracts of epidermis. Immunohistochemistry of vinculin in these tissues displayed two distinct locations for adherens junctions: i) at the dermal-epidermal junction, and ii) in the region of cell-cell contacts in all layers of the epidermis. The location of vinculin in the region of the epidermal-dermal junction is reminiscent of the distribution of vinculin-containing focal contacts in cultured keratinocytes, and the intercellular staining of vinculin in epidermis is consistent with the presence of vinculin in adherens junctions in cultured keratinocytes at sites of cell-cell contact. These results demonstrate that adherens junctions are present in human epidermis, oral mucosa, and monkey esophagus. Vinculin-containing junctions in epidermis may be important in the pathogenesis of skin diseases involving alterations in intercellular integrity.

Cell Adhesion↗