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K J Green

Publications and source records attributed to K J Green.

At least 19 recordsLinked to original sources
PubMed1992

Molecular structure of the human desmoplakin I and II amino terminus.

Desmoplakins (DPs) I and II are closely related proteins found in the innermost region of the desmosomal plaque, which serves as a cell surface attachment site for cytoplasmic intermediate filaments. Overlapping cDNA clones comprising 9.2 kilobases of DP-I, predicted to encode a full-length 310-kDa polypeptide (2677 amino acid residues), have now been identified. Here we report the predicted protein sequence and structural analysis of the N terminus of DP, extending our previous study of the rod and carboxyl domains. The N terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical-rich bundles. Unlike the rod and carboxyl domains, the N terminus did not display a periodic distribution of charged residues. Northern blot mapping and genomic sequence analysis were also undertaken to examine the organization of the DP mRNAs. A 1-kilobase intron was located at the 3' boundary of a DP-I-specific region; however, instead of an intron at the 5' junction, a possible splice donor site was observed within a potential coding sequence, suggesting alternative RNA splicing from an internal donor site.

Amino Acid Sequence↗
PubMed1992

Comparative structural analysis of desmoplakin, bullous pemphigoid antigen and plectin: members of a new gene family involved in organization of intermediate filaments.

Desmoplakins (DP) and bullous pemphigoid antigen (BPA) are major plaque components of the desmosome and hemidesmosome, respectively. These cell adhesion structures are both associated intimately with the intermediate filament (IF) network. Structural analyses of DP and BPA sequences have indicated that these molecules are likely to form extended dumbbell-shaped dimers with a central rod and globular end domains. Recent sequence data have indicated that the N-terminal domains of both DP and BPA (like their C-terminal domains) are highly related: the former contain regions of heptad repeats that are predicted to form several alpha-helical bundles. Comparisons of DP and BPA protein sequences with that of plectin (PL), a 466 kDa IF-associated protein, have also revealed large scale homology. Identities between their N-terminal domains are: DP:BPA = 35%, DP:PL = 32%, BPA:PL = 40%, suggesting that BPA is more closely related to PL than DP in this region. In the C-terminal domains, which contain a 38-residue repeating motif, however, DP and PL are closer relatives (identities: DP:BPA = 38%, BPA:PL = 40%, DP:PL = 49%). The central domains of all three proteins have extensive heptad repeat substructure, express the same periodic distribution of charged residues, and are predicted to form two-stranded alpha-helical coiled-coil ropes. These observations suggest that DP, BPA and PL belong to a new gene family encoding proteins involved in IF organization.

Amino Acid Sequence↗
PubMed1992

The desmoplakin carboxyl terminus coaligns with and specifically disrupts intermediate filament networks when expressed in cultured cells.

Specific interactions between desmoplakins I and II (DP I and II) and other desmosomal or cytoskeletal molecules have been difficult to determine in part because of the complexity and insolubility of the desmosome and its constituents. We have used a molecular genetic approach to investigate the role that DP I and II may play in the association of the desmosomal plaque with cytoplasmic intermediate filaments (IF). A series of mammalian expression vectors encoding specific predicted domains of DP I were transiently expressed in cultured cells that form (COS-7) and do not form (NIH-3T3) desmosomes. Sequence encoding a small antigenic peptide was added to the 3' end of each mutant DP cDNA to facilitate immunolocalization of mutant DP protein. Light and electron microscopical observations revealed that DP polypeptides including the 90-kD carboxy-terminal globular domain of DP I specifically colocalized with and ultimately resulted in the complete disruption of IF in both cell lines. This effect was specific for IF as microtubule and microfilament networks were unaltered. This effect was also specific for the carboxyl terminus of DP, as the expression of the 95-kD rod domain of DP I did not visibly alter IF networks. Immunogold localization of COS-7 cells transfected with constructs including the carboxyl terminus of DP demonstrated an accumulation of mutant protein in perinuclear aggregates within which IF subunits were sequestered. These results suggest a role for the DP carboxyl terminus in the attachment of IF to the desmosome in either a direct or indirect manner.

3T3 Cells↗
PubMed1992

The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the Drosophila segment polarity gene armadillo form a multigene family with similar properties.

Three proteins identified by quite different criteria in three different systems, the Drosophila segment polarity gene armadillo, the human desmosomal protein plakoglobin, and the Xenopus E-cadherin-associated protein beta-catenin, share amino acid sequence similarity. These findings raise questions about the relationship among the three molecules and their roles in different cell-cell adhesive junctions. We have found that antibodies against the Drosophila segment polarity gene armadillo cross react with a conserved vertebrate protein. This protein is membrane associated, probably via its interaction with a cadherin-like molecule. This cross-reacting protein is the cadherin-associated protein beta-catenin. Using anti-armadillo and antiplakoglobin antibodies, it was shown that beta-catenin and plakoglobin are distinct molecules, which can coexist in the same cell type. Plakoglobin interacts with the desmosomal glycoprotein desmoglein I, and weakly with E-cadherin. Although beta-catenin interacts tightly with E-cadherin, it does not seem to be associated with either desmoglein I or with isolated desmosomes. Anti-armadillo antibodies have been further used to determine the intracellular localization of beta-catenin, and to examine its tissue distribution. The implications of these results for the structure and function of different cell-cell adhesive junctions are discussed.

Amino Acid Sequence↗
PubMed1992

Structure of desmoplakin and its association with intermediate filaments.

Desmoplakins (DPs) I and II are two major related proteins located in the desmosomal plaque where they have been proposed to play a role in attaching intermediate filaments (IF) to the inner cell surface. The predicted amino acid sequence of DP was obtained by analysis of overlapping cDNA clones. Computer-aided analysis suggests that DPI will form a dumbbell-shaped homodimer, with a central alpha-helical coiled coil rod domain of 132 nm and two globular end domains. The DPII molecule is missing 599 residues from the central domain, resulting in a rod about one third the length of DPI. The carboxyl terminus comprises three subdomains each containing almost 5 repeats of a 38 residue repeating motif with a periodicity in acidic and basic residues similar to that found in the rod domain of IF proteins. This suggests a possible mechanism by which these proteins might interact. The amino terminus contains groups of heptad repeats that are predicted to form at least two major alpha-helical rich bundles. A series of c-myc-tagged mammalian expression vectors encoding specific predicted domains of DPI were transiently expressed in COS-7 cells. Light and electron microscopical observations revealed that DP polypeptides including the 90 kDa carboxyl terminal globular domain of DPI specifically colocalized with and ultimately resulted in the complete disruption of keratin and vimentin IF. This effect was specific for the carboxyl terminus, as the expression of the 95 kDa rod domain of DPI did not visibly alter IF networks.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Adhesion Molecules↗
PubMed1991

Desmoplakin expression and distribution in cultured rat bladder epithelial cells of varying tumorigenic potential.

The expression and distribution of the desmosomal plaque proteins, desmoplakins (DPs) I and II, were studied in nontumorigenic (RBE-8) and a series of tumorigenic (AY34, R-4909, SS-24B, RBTCC-8, and 804G) rat bladder epithelial cell lines. These cell lines ranged from slow-growing papillary transitional cells (AY34) to rapidly metastatic carcinoma cells (RBTCC-8). DPs I and II were shown by immunoblotting and Northern analysis to be present in nontumorigenic RBE-8 cells as well as in all of the tumorigenic cell lines, albeit in differing amounts. Immunofluorescence microscopy revealed striking differences in DP distribution, corresponding in general with increases in tumorigenic potential. Whereas DPs of normal RBE-8 cells and less tumorigenic AY34 cells were localized predominantly at cell interfaces, the more tumorigenic lines exhibited a high proportion of DP in the form of cytoplasmic dots, a distribution reminiscent of that seen in epithelial cells maintained in low levels of extracellular calcium. In 804G cells, which represented the most extreme example of this phenomenon, the majority of DPs were organized as cytoplasmic dots. Electron microscopy revealed intermediate filament (IF)-associated spots in the cytoplasm as well as an elaborate array of IF-associated plaques at the cell-substratum interface. The IF-associated spots in the cytoplasm reacted with anti-DP antibody in immunogold labeling experiments while those at the cell-substratum did not react. In more dense cultures of 804G cells, certain cells stratified and expressed increased amounts of DP followed by the induction of new keratins including those of the skin type. Decreasing extracellular calcium resulted in a rearrangement of DP in each cell line; staining at cell-cell interfaces disappeared and was replaced with a pattern of cytoplasmic dots. These results demonstrate a possible relationship between desmosome assembly and/or maintenance and tumorigenic potential.

Animals↗
PubMed1991

Intermediate filament-plasma membrane interactions.

In this review we will discuss the molecules involved in intermediate filament-desmosome and intermediate filament-hemidesmosome interactions, and the means by which certain of these molecules may bind to intermediate filaments. The possibility that intermediate filaments interact directly with peripheral membrane proteins and membrane lipids will also be addressed.

Amino Acid Sequence↗
PubMed1991

Formation of hemidesmosomes in vitro by a transformed rat bladder cell line.

Two hemidesmosomal plaque components of 230 and 180 kD have recently been characterized using autoantibodies in the serum samples of bullous pemphigoid (BP) patients (Klatte, D. H., M. A. Kurpakus, K. A. Grelling, and J. C. R. Jones. 1989, J. Cell Biol. 109:3377-3390). These BP autoantibodies generate the type of staining patterns that one would predict for formed hemidesmosomes, i.e., a punctate staining pattern towards the substratum; in less than 50% of various primary epithelial and transformed epidermal cell lines even when such cells are maintained in culture for prolonged periods. In contrast, affinity-purified human autoantibodies against the 230-kD hemidesmosomal plaque component generate intense immunofluorescence staining along the region of cell-substratum interaction in the rat bladder tumor cell line 804G maintained on uncoated glass cover-slips. This pattern is distinct from that observed in the 804G cells using an antibody preparation directed against vinculin, a component of adhesion plaques. Ultrastructural analyses of the 804G cells reveals that hemidesmosome-like structures occur along the basal surface of cells where they abut the substratum. These structures are present in 804G cells maintained in culture in reduced levels of Ca2+ and are recognized by autoantibodies directed against the 230-kD hemidesmosomal plaque component as determined by immunogold ultrastructural localization. To study hemidesmosome appearance in this cell line, 804G cells were trypsinized and then allowed to readhere to glass coverslips. In rounded, unattached 804G cells, hemidesmosome-like plaque structures occur along the cell surface. These structures are recognized by the 230-kD autoantibodies. At 1 h after plating, hemidesmosomes are observed along the substratum attached surface of cells. Protein synthesis is not required for the appearance of these hemidesmosomes. Within 4 h of plating, autoantibody staining and hemidesmosomes appear towards the cell periphery. Subsequently, the polypeptide recognized by the BP autoantibodies becomes concentrated in the perinuclear region, where there are numerous hemidesmosomes. We propose that the hemidesmosomes in 804G cells are involved in cell-substratum adhesion. We discuss possible mechanisms of assembly of hemidesmosomes in the 804G cells. Indeed, the 804G cells should prove an invaluable cell line for the biochemical and molecular dissection of hemidesmosome structure, function, and assembly.

Animals↗
PubMed1991

Structural analysis and expression of human desmoglein: a cadherin-like component of the desmosome.

Desmosomes are adhesive cell junctions found in great abundance in tissues that experience mechanical stress. The transmembrane desmosomal glycoproteins have been proposed to play a role in cell adhesion; desmoglein I (DGI) is a major member of this class of desmosomal molecules. However, evidence supporting a role for DGI in cell adhesion or in the plaque is lacking. In order to begin to understand DGI function we have identified human cDNA clones encoding the entire mature polypeptide of 1000 amino acids. Our data suggest that like the bovine DGI molecule human DGI is highly related to the calcium-dependent class of cell adhesion molecules known as cadherins. Four related extracellular domains located in the amino-terminal domain of the molecule contain putative calcium binding sites originally identified in the cadherins. The highest degree of similarity between human N-cadherin and human DGI, and likewise between bovine DGI and human DGI, is greatest in the most amino-terminal extracellular domain. This suggests a conserved functional role for the extracellular domains, perhaps in calcium-mediated cell adhesion. The cytoplasmic portion of the molecule contains a cadherin-like region and, like bovine DGI, a carboxy-terminal tail that is not present in the cadherins, comprising three additional domains. One of these contains a novel repeating motif of 29 +/- 1 residues, first identified in bovine DGI. Each of the highly homologous repeating units is likely to consist of two beta-strands and two turns with special characteristics. Five amino acids that are identical in bovine and human DGI lie in the second of the two predicted beta-strands, and intriguingly contain putative target sites for protein kinase C. On the basis of structural analysis, a model predicting the disposition of human DGI domains in the desmosome is proposed. Northern analysis suggests that unlike bovine epidermis, which expresses a single mRNA of reported size approximately 7.6 kb, human foreskin and cultured keratinocytes display a complex pattern with bands of approximately 7.2, 4.0 and 3.0 kb. Each of these cross-hybridizing mRNAs is coordinately expressed in normal human keratinocytes in response to long-term culture and increased calcium.

Amino Acid Sequence↗
PubMed1990

Structure of the human desmoplakins. Implications for function in the desmosomal plaque.

Desmoplakins (DPs) I and II are two major related proteins located in the innermost portion of the desmosomal plaque where it is thought they may play a role in attaching intermediate filaments (IF) to the cell surface. We have isolated and sequenced human cDNA clones encoding two major DP domains and a portion of a third. These clones can be divided into two classes that we believe to represent DPI and DPII cDNAs; our evidence suggests that the DPII message is derived at least in part from the processing of a larger transcript encoded by a single gene. Computer-aided analysis of the DPI-predicted amino acid sequence indicates that the central domain, which contains the heptad repeat characteristic of many alpha-fibrous proteins, will participate in the formation of a coiled coil dimer approximately 130 nm in length. The periodicity of acidic and basic residues in the rod suggests that DPI will aggregate with itself or similar molecules into higher order filamentous structures. The carboxyl terminus contains three regions with significant homology, each of which comprises almost five repeats of a 38-residue motif. It is likely that these regions each fold into a compact globular conformation stabilized by intrachain ionic interactions. Comparison of the predicted amino acid sequence of a cDNA encoding a portion of the 230-kDa bullous pemphigoid antigen (Stanley, J. R., Tanaka, T., Mueller, S., Klaus-Kovtun, V., and Roop, D. (1988) J. Clin. Invest. 82, 1864-1870) with DP revealed the presence of a 38-residue repeat with striking similarity to that of the DPs. Significantly, the periodicity in acidic and basic residues of these domains is the same as that found in the 1B rod domain of IF proteins. This suggests the possibility that the DPs might interact with IF via their common periodicity of charged residues.

Amino Acid Sequence↗
PubMed1990

Desmoplakin II expression is not restricted to stratified epithelia.

Desmosomes are major intercellular junctions found in association with intermediate filaments in epithelial, cardiac and arachnoidal tissue. Desmoplakins I and II (DPI and II) are highly related proteins localized in the innermost part of the desmosomal plaque and are candidates for linking intermediate filaments (IF) to the desmosomal complex. While investigators agree that DPI is present in all epithelia, they disagree on the distribution of DPII. Some have reported DPII to be restricted to stratified tissue and have furthermore suggested that the expression of DPII may be linked to stratification. We have compared the expression of DPI and II at the mRNA and protein levels in cell lines derived from simple, transitional and stratified epithelia. Northern blot analysis revealed DPI and II mRNA to be present in all cell lines as well as simple and stratified epithelial tissues. However, DPII mRNA could not be detected in cardiac muscle tissue. Immunoblotting and immunoprecipitation demonstrated the presence of DPI and II in all cell lines at the whole-cell protein level as well as in association with cytoskeletal fractions. Immunofluorescence staining was used to correlate the biochemical findings with the localization of DPI and II. While most cell lines exhibited typical intercellular and in many cases cytoplasmic DP staining, T24 cells exhibited predominantly diffuse and dotty cytoplasmic staining. In addition, we investigated whether changes in DPI and II expression occurred following calcium-induced cell contact formation and stratification in the human pharyngeal cell line, FaDu. No significant changes in mRNA or whole-cell protein levels were observed during a period of 5 days following the calcium switch. However, immunoblotting revealed a significant increase in DPI and II levels in the insoluble protein pool during desmosome formation. These observations indicated a possible recruitment of soluble DPI/II into an insoluble pool after induction of desmosome assembly by the calcium switch, consistent with earlier reports for MDCK cells. In summary, our results suggest that the expression of DPII is not strictly linked to stratification or differentiation; however, the apparent absence of DPII mRNA from cardiac muscle suggests it may not be a constituent of all desmosomes.

Calcium↗
PubMed1988

Isolation of cDNAs encoding desmosomal plaque proteins: evidence that bovine desmoplakins I and II are derived from two mRNAs and a single gene.

Desmoplakins (DPs) I and II (approximately equal to 240 and approximately equal to 210 kDa) are major components of the internal portion of the desmosomal cytoplasmic plaque. Desmosomes play a crucial role in cell-cell adhesion and serve as specific attachment sites for cytoplasmic intermediate filaments. Although DP-I and -II are closely related molecules, their structure (i.e., amino acid or DNA sequence) has not been determined. In addition, it is not known whether these proteins are derived from one or more genes or whether they result from posttranscriptional or posttranslational events. This paper describes the isolation and characterization of eight DP cDNA clones from a bovine lambda gt11 expression library. Fusion proteins from six of these clones selected antibodies that reacted with DP-I and -II and two selected antibodies that reacted with DP-I alone. Antibodies made against fusion protein produced by the DP1A clone reacted specifically with DP-I and -II on immunoblots. When used for indirect immunofluorescence on bovine tongue cryostat sections and cultured mouse keratinocytes, these antibodies produced a typical desmosomal staining pattern. RNA blot analysis demonstrated hybridization of three DP-I/II cDNA probes with two messages of approximately equal to 7.5 and approximately equal to 9.5 kilobases in bovine tongue RNA. In contrast, a cDNA clone that affinity-purified antibodies reacting with DP-I only hybridized exclusively with the 9.5-kilobase band. Southern blots of genomic DNA digested with a panel of restriction enzymes were hybridized with one probe derived from a DP-I/II clone and with one from a DP-I clone. Both probes hybridized with single bands of the same size in each digested sample of DNA. Together, these data suggest that DP-I and DP-II are translated from two separate messages in bovine tongue and that these messages may be derived from a single gene.

Animals↗
PubMed1987

Results of screening a large group of intercollegiate competitive athletes for cardiovascular disease.

To determine the feasibility of detecting cardiovascular disease in a large group of young competitive athletes, a prospective screening evaluation of intercollegiate student athletes was undertaken at the University of Maryland. Initial clinical screening (including personal and family history, physical examination and 12 lead electrocardiogram) was performed in 501 athletes. Ninety of these subjects had positive findings on one or more of the three studies and agreed to further cardiologic evaluation. The vast majority (75 [84%] of 90) had no definitive evidence of cardiovascular disease, although 1 athlete had mild systemic hypertension and 14 (15%) had echocardiographic evidence of relatively mild mitral valve prolapse that had not been previously suspected. In three athletes with relatively mild ventricular septal hypertrophy (14 to 15 mm), it was not possible to discern with absolute certainty whether the wall thickening was a manifestation of hypertrophic cardiomyopathy or secondary to athletic conditioning ("athlete heart"). Therefore, this screening protocol identified no athletes with definite evidence of hypertrophic cardiomyopathy, Marfan's syndrome or other cardiovascular diseases that convey a significant potential risk for sudden death or disease progression during athletic activity. This failure to identify such diseases could have been due to a lack of sensitivity of the screening tests or to the low frequency with which these diseases occur in youthful healthy athletes. A systematic preparticipation screening program (such as the present one) does not appear to be an efficient means of detecting clinically important cardiovascular disease in young athletes.

Adolescent↗
PubMed1987

Noninvasive assessment of left ventricular diastolic function by pulsed Doppler echocardiography in patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is a primary myocardial disease in which symptoms may frequently result from impaired left ventricular relaxation, filling and compliance. In the present investigation, Doppler echocardiography was utilized to measure transmitral flow velocity and thereby assess left ventricular diastolic performance noninvasively in a group of 111 patients representative of the broad clinical spectrum of hypertrophic cardiomyopathy. In patients with hypertrophic cardiomyopathy, all Doppler indexes of diastolic relaxation and filling differed significantly (p less than 0.001) from those obtained in 86 control subjects without heart disease, namely, prolongation of isovolumic relaxation (94 +/- 24 versus 78 +/- 12 ms) and of the early diastolic peak of flow velocity (244 +/- 55 versus 220 +/- 28 ms), as well as slower deceleration (3.4 +/- 1.4 versus 4.9 +/- 1.3 m/s2) and reduced maximal flow velocity in early diastole (0.5 +/- 0.2 versus 0.6 +/- 0.1 m/s). As an apparent compensation for impaired relaxation and early diastolic filling, the atrial contribution to left ventricular filling was increased, as shown by increased late diastolic flow velocity (0.4 +/- 0.3 versus 0.3 +/- 0.1 m/s) and reduced ratio of maximal flow velocity in early diastole to that in late diastole (1.4 +/- 0.8 versus 2.1 +/- 0.9). The vast majority of patients with hypertrophic cardiomyopathy (91 [82%] of 111) showed evidence of impaired left ventricular diastolic performance, as assessed from the Doppler waveform. Abnormal Doppler diastolic indexes were identified with similar frequency in patients with (78%) or without (83%) left ventricular outflow obstruction, as well as in patients with (84%) or without (80%) cardiac symptoms. However, patients with nonobstructive hypertrophic cardiomyopathy showed more severe alterations in the Doppler indexes of diastolic function than did patients with obstruction. Thus, abnormal diastolic performance as assessed by Doppler echocardiography was apparent in the vast majority of the study patients with hypertrophic cardiomyopathy, independent of the presence or absence of cardiac symptoms or a subaortic pressure gradient. The high frequency with which diastolic abnormalities are identified in asymptomatic patients with hypertrophic cardiomyopathy suggests that impaired diastolic performance may be present at a time in the natural history of the disease when functional limitation is not yet evident.

Adolescent↗
PubMed1987

The relationship between intermediate filaments and microfilaments before and during the formation of desmosomes and adherens-type junctions in mouse epidermal keratinocytes.

Actin, keratin, vinculin and desmoplakin organization were studied in primary mouse keratinocytes before and during Ca2+-induced cell contact formation. Double-label fluorescence shows that in cells cultured in low Ca2+ medium, keratin-containing intermediate filament bundles (IFB) and desmoplakin-containing spots are both concentrated towards the cell center in a region bounded by a series of concentric microfilament bundles (MFB). Within 5-30 min after raising Ca2+ levels, a discontinuous actin/vinculin-rich, submembranous zone of fluorescence appears at cell-cell interfaces. This zone is usually associated with short, perpendicular MFB, which become wider and longer with time. Later, IFB and the desmoplakin spots are seen aligned along the perpendicular MFB as they become redistributed to cell-cell interfaces where desmosomes form. Ultrastructural analysis confirms that before the Ca2+ switch, IFB and desmosomal components are found predominantly within the perimeter defined by the outermost of the concentric MFB. Individual IF often splay out, becoming interwoven into these MFB in the region of cell-substrate contact. In the first 30 min after the Ca2+ switch, areas of submembranous dense material (identified as adherens junctions), which are associated with the perpendicular MFB, can be seen at newly formed cell-cell contact sites. By 1-2 h, IFB-desmosomal component complexes are aligned with the perpendicular MFB as the complexes become redistributed to cell-cell interfaces. Cytochalasin D treatment causes the redistribution of actin into numerous patches; keratin-containing IFB undergo a concomitant redistribution, forming foci that coincide with the actin-containing aggregates. These results are consistent with an IF-MF association before and during desmosome formation in the primary mouse epidermal keratinocyte culture system, and with the temporal and spatial coordination of desmosome and adherens junction formation.

Actin Cytoskeleton↗
PubMed1986

Variations in flow-velocity waveforms obtained by pulsed Doppler echocardiography in the normal human aorta.

To characterize the contour and duration of aortic flow-velocity waveforms in the normal human, the ascending aorta of 23 persons without evidence of cardiovascular disease was examined systematically with pulsed Doppler echocardiography. In 16 of the 23 subjects, measurements throughout the ascending aorta showed flow-velocity waveforms of similar contour and duration, characterized by flow-velocity peaking in early to midsystole, with most flow velocity (60 +/- 4%) occurring in the first half of the available systolic ejection period, and then gradually decreasing to 0 baseline coincident with aortic valve closure. In the other 7 subjects, aortic flow-velocity waveforms recorded at most of the sampling sites also revealed a normal flow-velocity pattern; however, in each of these subjects, 1 to 3 sites that displayed a distinct alteration from the normal pattern were also identified. The waveforms recorded from these latter sites were characterized by flow-velocity peaking earlier in systole and decelerating to 0 baseline approximately 100 ms before aortic valve closure; consequently, a particularly large fraction of flow velocity (88 +/- 9%) occurred in the first half of the systolic ejection period. These apparently shortened waveforms were always detected at sites near the medial aortic wall and often at or near the junction of the ascending and transverse aorta. Hence, aortic flow-velocity waveforms with altered contour and duration (resembling those recorded in patients with obstructive hypertrophic cardiomyopathy) were infrequently identified by pulsed Doppler echocardiography in subjects with normal hearts and were not characteristic of the overall aortic flow-velocity pattern in any of these subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1986

Evidence for an interaction between the cell surface and intermediate filaments in cultured fibroblasts.

Intermediate filaments (IF) were found in close proximity to the plasma membrane in substrate attached baby hamster kidney cells (BHK-21) and chick embryo fibroblasts (CEF) as well as cells removed from their substrate in the absence of trypsin. However, in cells removed with trypsin, it appeared that IF had retracted away from the membrane. In cells with abundant extracellular matrix (ECM), colchicine induced massive cables of IF, which appeared to interact with specialized areas of the inner plasma membrane. In cells lysed to extract most microfilaments and cytoplasmic constituents, the intact IF network which remained was closely associated with the ECM. From these ultrastructural observations it was concluded that IF interact in some way with a "cell membrane complex" defined as comprising the plasma membrane and molecules attached to its inner and outer surfaces. In order to investigate the possibility that components of the membrane complex may co-isolate with IF, native intermediate filaments (NIF) were prepared. In addition to the structural subunits and other associated polypeptides, a approximately 220 kd species which reacted specifically with antibodies directed against the ECM protein fibronectin (FN) was observed; 220 kd was still present after NIF were isolated under pH conditions where FN is more soluble, suggesting that its presence was not simply due to the coprecipitation of two insoluble proteins. Immunofluorescence and immunogold localization confirmed that FN is a component of the cell membrane complex with which IF appeared to interact.

Animals↗