Biomedical subjects

M R Block

Publications and source records attributed to M R Block.

At least 19 recordsLinked to original sources

PubMed2004

Early enterocytic differentiation of HT-29 cells: biochemical changes and strength increases of adherens junctions.

We have characterized the modulation of cell-cell adhesion and the structure of adherens junctions in the human colon adenocarcinoma HT-29 cell line that differentiates into enterocytes after glucose substitution for galactose in the medium. We demonstrate that differentiated cells (HT-29 Gal) rapidly established E-cadherin-mediated interactions in aggregation assays. This effect is not due to an increase in E-cadherin expression during this early stage of cell differentiation, but rather results from the maturation of preexisting adherens junctions. These junctions are characterized by the redistribution of E-cadherin to the basolateral membrane and its co-localization with the actin cytoskeleton. Subcellular fractionation studies indicate that actin-associated E-cadherins bind beta-catenin and p120ctn. Furthermore, the p120ctn/E-cadherin association is upregulated. These data reveal a cooperative interaction between p120ctn and E-cadherin that corresponds to mature functional adherens junctions able to initiate tight cell-cell adhesion required for epithelium architecture and further affirm the gatekeeper role of p120ctn.

Actins↗

PubMed2001

Conformation, localization, and integrin binding of talin depend on its interaction with phosphoinositides.

Talin is a structural component of focal adhesion sites and is thought to be engaged in multiple protein interactions at the cytoplasmic face of cell/matrix contacts. Talin is a major link between integrin and the actin cytoskeleton and was shown to play an important role in focal adhesion assembly. Consistent with the view that talin must be activated at these sites, we found that phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) bound to talin in cells in suspension or at early stages of adhesion, respectively. When phosphoinositides were associated with phospholipid bilayer, talin/phosphoinositide association was restricted to PI4,5P(2). This association led to a conformational change of the protein. Moreover, the interaction between integrin and talin was greatly enhanced by PI4,5P(2)-induced talin activation. Finally, sequestration of PI4,5P(2) by a specific pleckstrin homology domain confirms that PI4,5P(2) is necessary for proper membrane localization of talin and that this localization is essential for the maintenance of focal adhesions. Our results support a model in which PI4,5P(2) exposes the integrin-binding site on talin. We propose that PI4,5P(2)-dependent signaling modulates assembly of focal adhesions by regulating integrin-talin complexes. These results demonstrate that activation of the integrin-binding activity of talin requires not only integrin engagement to the extracellular matrix but also the binding of PI4,5P(2) to talin, suggesting a possible role of lipid metabolism in organizing the sequential assembly of focal adhesion components.

3T3 Cells↗

PubMed2001

RhoA-dependent switch between alpha2beta1 and alpha3beta1 integrins is induced by laminin-5 during early stage of HT-29 cell differentiation.

Integrin-mediated interactions between the basement membrane and epithelial cells control the differentiation of epithelia. We characterized the modulation of adhesive behaviors to basement membrane proteins and of integrin function in the human colon adenocarcinoma HT-29 cell line, which differentiates into enterocytes after the substitution of galactose for glucose in the medium. We demonstrate an increased capability of these cells to adhere to collagen type IV during the early stage of differentiation. This effect occurs without any changes in integrin cell surface expression but rather results from an alpha2beta1/alpha3beta1 integrin switch, alpha3beta1 integrin becoming the major collagen receptor. The increase in laminin-5 secretion and deposit on the matrix is a key factor in the mechanism regulating cell adhesion, because it is responsible for the activation of alpha3beta1 integrin. Furthermore, down-regulation of RhoA GTPase activity occurs during HT-29 cell differentiation and correlates with the activation of the integrin alpha3beta1. Indeed, C3 transferase, a RhoA GTPase inhibitor, induces a similar alpha2beta1/alpha3beta1 switch in undifferentiated HT-29 cells. These results indicate that the decrease in RhoA activation is the biochemical mechanism underlying this integrin switch observed during cell differentiation. The physiological relevance of such modulation of integrin activity in the functioning of the crypt-villus axis is discussed.

Cell Adhesion↗

PubMed2000

Talin controls the exit of the integrin alpha 5 beta 1 from an early compartment of the secretory pathway.

Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.

Antisense Elements (Genetics)↗

PubMed2000

Adherence of human erythroleukemia cells inhibits proliferation without inducing differentiation.

To investigate the effect of extracellular matrix molecules in the megakaryocytic lineage, we studied the role of integrin engagement in the proliferation and differentiation of human erythroleukemia (HEL) cells. HEL cells grew in suspension, but their adherence depended upon the presence of matrix proteins or protein kinase C signaling. Adherence by itself did not trigger commitment of these cells but accelerated phorbol 12-myristate 13-acetate-induced differentiation. HEL cells adhered to fibronectin mainly through alpha5beta1, and this receptor acted synergetically with alpha4beta1. Integrin engagement induced cell growth arrest through mitogen-activated protein kinase inactivation. Such down-regulation of the mitogen-activated protein kinase pathway by integrin engagement was suggested as a megakaryocytic-platelet lineage specificity. This signaling was not restricted to a peculiar integrin but was proposed as a general mechanism in these cells.

Cell Adhesion↗

PubMed1999

Grafting an RGD motif onto an epidermal growth factor-like module: chemical synthesis and functional characterization of the chimeric molecule.

A novel protein was engineered by inserting the GRGDS motif of fibronectin within the 14-residue loop of the EGF-like module from human complement protease C1r. The resulting chimeric EGF-RGD module (52 residues, three disulfide bridges) was assembled by automated solid-phase synthesis using the t-Boc strategy. Using reduced/oxidized glutathione, the EGF-RGD module was folded as efficiently as the natural C1r-EGF module, resulting in formation of the appropriate disulfide bridge pattern as shown by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. Circular dichroism and NMR measurements provided further indication that introduction of the GRGDS motif had no significant effect on the folding. Using Chinese Hamster Ovary (CHO) cells bearing the integrin receptors specific for fibronectin and vitronectin, EGF-RGD was shown to induce cell adhesion via the introduced GRGDS motif. Cell binding was inhibited specifically and efficiently by the synthetic peptide GRGDSP and by fibronectin, and to a much lesser extent by vitronectin, whereas the monoclonal antibody PB1 directed to the alpha5 subunit of alpha5beta1 integrin had no effect. The ability of EGF-RGD to trigger significant cell spreading and intracellular signaling was also demonstrated using immunofluorescence and confocal microscopy.

Amino Acid Sequence↗

PubMed1998

Calcium/calmodulin-dependent protein kinase II controls integrin alpha5beta1-mediated cell adhesion through the integrin cytoplasmic domain associated protein-1alpha.

This paper provided evidence that the regulation of CHO cell adhesion on fibronectin by calcium/calmodulin-dependent protein kinase II (CaMKII) is mediated through the recently described integrin cytoplasmic domain associated protein-1alpha (ICAP-1alpha). The point mutation T38D localized within the optimal CaMKII recognition motif of ICAP-1alpha results in a strong defect in cell spreading which cannot be overcome by the inhibition of the endogenous CaMKII. This fact strongly suggests that the phosphorylation of Threonine 38 by CaMKII modulates the alpha5beta1 integrin function. Conversely, the mutation T38A produces an analog of ICAP-1alpha that cannot be phosphorylated and that stimulates cell spreading on fibronectin to a similar extent when CaMKII is inhibited.

Adaptor Proteins, Signal Transducing↗

PubMed1998

Adhesion of mature polyploid megakaryocytes to fibronectin is mediated by beta 1 integrins and leads to cell damage.

Human CD34+ bone marrow cells were committed to the megakaryocytic lineage in serum-free liquid cultures by the following cytokines: thrombopoietin, erythropoietin, and IL-6. Megakaryocyte maturation has been described as being regulated by the extracellular matrix. These cells express receptors for laminin, collagen, and vitronectin, but they selectively adhere to and spread on fibronectin, a major component of the bone marrow environment. Function-perturbing antibodies against beta 1 integrins totally abolished the adhesion of megakaryocytes on fibronectin, whereas antibodies to beta 3 did not, suggesting that beta 1 integrins were responsible for the adhesive phenotype of these polyploid cells. beta 1-positive clusters were visualized in close contact with the extremities of stress fibers at the cell surface. In the course of cell spreading, we observed morphological modifications such as the disorganization of the compact nuclei structure and the appearance of holes in the cytoplasm leading to the release of alpha IIb beta 3-positive cellular fragments. This process appeared to be a specific feature of megakaryocytes and is correlated neither to apoptosis nor to integrin signaling.

Actin Cytoskeleton↗

PubMed1998

Calcium/calmodulin-dependent protein kinase II controls alpha5beta1 integrin-mediated inside-out signaling.

Fibronectin binding on alpha5beta1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of alpha5beta1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited alpha5beta1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 microM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in alpha5beta1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin alpha5beta1 in vitro and in living cells.

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

PubMed1997

Treating depressed primary care patients improves their physical, mental, and social functioning.

BACKGROUND: This study describes the functioning of primary care patients with major depressive disorder, the relationship of medical comorbidity to functional status, and the effects of depression-specific treatment on functional status after 8 months. METHODS: Patients were randomized to a protocol intervention (nortriptyline hydrochloride or interpersonal psychotherapy) or to usual care with the patient's physician in a clinical trial of primary care treatments of depression. Their functional status was evaluated using the Medical Outcomes Study 36-Item Short Form Health Survey (SF-36) and the Global Assessment Scale. Medical comorbidity was assessed with the Duke Severity of Illness Checklist. The Hamilton Rating Scale for Depression and Beck Depression Inventory were used to measure depressive severity. Assessments were conducted at baseline and at 1, 2, 4, and 8 months after randomization. RESULTS: At baseline, patients reported substantial impairments in the functional domains as assessed by the SF-36 and Global Assessment Scale. Severity of general medical illness and depression were not correlated. Greater medical comorbidity was associated with diminished physical, but not psychological, functioning. Mean scores on SF-36 scales and the Global Assessment Scale improved significantly during the 8 months of follow-up. Patients assigned to protocol treatments showed greater improvement, compared with those assigned to usual care, on the SF-36 mental summary scale and most individual scales but not on the SF-36 physical summary scale. However, patients who completed protocol treatment also experienced significant improvement on the physical summary scale. Medical comorbidity was only a weak predictor of outcome. CONCLUSIONS: Primary care patients with major depressive disorder report substantial impairments in physical, psychological, and social functioning on initial assessment. Severity of baseline medical comorbidity did not correlate with severity of depression and only weakly correlated with functional status at 8 months. Functional impairments improve with time, but standardized depression-specific treatment is associated with greater improvement in more domains of functioning than is a physician's usual care.

Adolescent↗

PubMed1997

Symptoms of major depression and tricyclic side effects in primary care patients.

OBJECTIVE: To examine the prevalence and course of symptoms resembling side effects of tricyclic antidepressants among primary care patients experiencing major depression and receiving nortriptyline pharmacotherapy. DESIGN: Prospective cohort study. PATIENTS: Seventy-five patients meeting DSM-III-R criteria for a current major depression. SETTING: Four Pittsburgh (Pa.) ambulatory health centers affiliated with residency programs. MEASUREMENTS AND MAIN RESULTS: Symptoms resembling tricyclic side effects were assessed at baseline and at monthly intervals using the Somatic Symptoms Checklist. The Hamilton Rating Scale for Depression and Diagnostic Interview Schedule were used to assess depressive severity and history of generalized anxiety or panic disorder, respectively. Symptoms resembling tricyclic side effects, including thirst (54%), palpitations (51%), and dry mouth (48%), were commonly experienced before commencing pharmacotherapy. Patients with severe depressive episodes and those with a history of an anxiety or panic disorder had significantly more physical symptoms than those with milder episodes of depression and were more likely to drop out of care (n = 25) before completing the acute phase of pharmacotherapy. Patients who completed the acute phase of pharmacotherapy and those who entered its continuation phase (n = 43) experienced significant reduction in many depressive and physical symptoms (p < .001). CONCLUSIONS: Symptoms resembling tricyclic side effects are common among depressed primary care patients before beginning pharmacotherapy and generally remit with the depressive episode. Better awareness of major depression's somatic effects and the consequences of therapy could result in better management of both physicians' and patients' expectations regarding antidepressant pharmacotherapy.

Adolescent↗

PubMed1997

NPXY motifs control the recruitment of the alpha5beta1 integrin in focal adhesions independently of the association of talin with the beta1 chain.

With the exception of the divergent beta4 and beta8 chains, the integrin beta subunit cytoplasmic domains are short and highly conserved sequences. Consensus motifs are found among the different cytoplasmic beta chains. Experiments using chimeric receptors demonstrated that the 47 amino acids of the beta1 subunit cytoplasmic domain contain sufficient information to target integrins to adhesion plaques. Three clusters of amino acids, named cyto-1, cyto-2 and cyto-3, seem to contribute to this localization. Cyto-2 and cyto-3 exhibit NPXY motifs. At present, the exact function of these motifs remains unknown but it is likely that these sequences are involved in protein-protein interactions. Although NPXY motifs often act as internalization signals at the cytoplasmic tail of membrane receptors, our previous results showed that the two NPXY motifs are not responsible for the alpha5beta1 integrin endocytosis. Herein, we address the question of the role of the two highly conserved NPXY motifs found in the beta1 cytoplasmic domain, and which correspond to the conserved domains cyto-2 and cyto-3. We demonstrate that, within the integrin beta1 cytoplasmic tail, the two NPXY motifs are required for the recruitment of the integrin in focal adhesions. In addition, our results indicate that these two motifs control but do not belong to the talin-binding sites. Finally, the analysis of the phenotypes of NPXY mutants reveals that the interaction of talin with the beta1 cytosolic domain is not sufficient to target the integrins to focal adhesions.

Amino Acid Sequence↗

PubMed1996

Cotranscription of two RNA coding for the cell adhesion regulator and its variant in Reh leukemia cells.

Human genomic DNA analysis reveals the existence of polymorphisms at the cell molecular adhesion regulator (CMAR) locus. In order to choose between the two possible open frames deduced from the variant sequence, we have sequence both the human 5' non-coding region and the mouse CMAR variant DNA. We found that both mRNA species coexist in human cells.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1996

Treating major depression in primary care practice. Eight-month clinical outcomes.

BACKGROUND: We studied whether standardized treatments of major depression whose efficacy was established with psychiatric patients are equally effective when provided to primary care patients, and whether standardized treatments are more effective than a primary care physician's usual care. METHODS: A randomized controlled trial was conducted, in which primary care patients meeting DSM-III-R criteria for a current major depression were assigned to nortriptyline (n = 91) or interpersonal psychotherapy (n = 93) provided within well-structured parameters, or a physician's usual care (n = 92). The main outcome measures were degree and rate of improvement in severity of depressive symptoms and proportion of patients recovered at 8 months. RESULTS: Severity of depressive symptoms was reduced more rapidly and more effectively among patients randomized to pharmacotherapy or psychotherapy than among patients assigned to a physician's usual care. Among treatment completers, approximately 70% of patients participating in the full pharmacotherapy or psychotherapy protocol but only 20% of usual care patients were judged as recovered at 8 months. CONCLUSIONS: Pharmacotherapy and psychotherapy effectively treat major depression among primary care patients when provided within specific parameters and for the full acute and continuation phases. Treatment principles recommended by the Depression Guideline Panel of the Agency for Health Care Policy and Research are supported.

Adult↗

PubMed1995

Control of the alpha 5 beta 1 integrin/fibronectin interaction in vitro by the serine/threonine protein phosphatase calcineurin.

Using Chinese hamster ovary cell lysate, an in vitro assay has been developed to study the interaction of fibronectin with the alpha 5 beta 1 integrin in a cytosolic environment. In our solid phase assay, 96-well microtiter plates were coated with fibronectin in which cell lysate was incubated. A dose-dependent binding of the fibronectin receptor onto the coated plastic was immunodetected by specific polyclonal antibodies raised against the alpha 5 beta 1 integrin. Both soluble fibronectin and PB1, a monoclonal antibody raised against the fibronectin receptor, competed with the alpha 5 beta 1 integrin for binding to the fibronectin-coated plastic. General phosphatase inhibitors used during cell lysis completely abolished the fibronectin/integrin interaction in the assay, indicating that the affinity of the fibronectin receptor might be modulated by a protein phosphatase activity. Furthermore, in this assay, the interaction between the fibronectin receptor and its substrate in a cytosolic environment required intracellular calcium. Additionally, the action of more specific phosphatase inhibitors and the inhibition of the integrin/fibronectin interaction by a monoclonal antibody raised against the calcium/calmodulin-dependent protein phosphatase calcineurin suggested that calcineurin allowed the interaction between the alpha 5 beta 1 integrin and fibronectin. Metabolical labeling experiments showed that alpha 5 beta 1 itself was not the target of phosphorylation/dephosphorylation cascades involving calcineurin and leading to the modulation of integrin affinity. Taken together, these results showed that in vitro one substrate of the serine/threonine protein phosphatase calcineurin regulates the alpha 5 beta 1 integrin affinity by interacting with a yet unidentified effector.

Animals↗

PubMed1995

Applicability of clinical pharmacotherapy guidelines for major depression in primary care settings.

OBJECTIVE: To determine whether guidelines established for pharmacologic treatment of major depression are feasible in primary care. DESIGN: Prospective cohort study. SETTING: Ambulatory family health centers and internal medicine clinics. PATIENTS: Ninety-one primary care patients meeting criteria within the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition for a current major depression randomized to receive antidepressant medication after being judged by a psychiatrist as clinically eligible for pharmacotherapy in an ambulatory setting. INTERVENTION: Nortriptyline hydrochloride prescribed by primary care physicians trained in clinical guidelines specifying dosage schedules, durations, and procedures resembling those recommended by the AHCPR (Agency for Health Care Policy and Research) Depression Guideline Panel. MAIN OUTCOME MEASURES: Patient participation and continuation in medication treatment. RESULTS: Fifty-five percent of patients completed the acute phase of treatment after a mean of 6.9 visits extending over a mean of 8.1 weeks. Of those patients entering the continuation phase, 60% completed the follow-up visits for 6 months. Taken together, only 33% of patients assigned to receive antidepressant medication completed the full regimen recommended by the AHCPR guidelines. CONCLUSIONS: The treatment of depressed primary care patients within AHCPR guidelines for antidepressant medication is feasible but complex. Although primary care physicians ably adhere to these guidelines, keeping patients in treatment is difficult and possibly requires greater flexibility in treatment regimens.

Adult↗

PubMed1995

Intracellular processing of talin occurs within focal adhesions.

CHO cells spread out on fibronectin-coated plastic were perforated with the bacterial toxin alveolysin. This treatment preserves the integrity of the cells but opens large and stable hydrophilic pores on the plasma membrane. With these semi-intact cells, it has been possible to have a direct access to adhesion plaques. Furthermore, with this procedure one can determine the distribution of a single intracellular protein between membrane-associated and cytosolic pools. The introduction within the perforated cells of polyclonal antibodies raised against talin induced the detachment of the cells. This provides direct evidence that talin is required to stabilize the adhesion plaques. Furthermore, immunoprecipitation of talin in the membrane-associated and cytosolic fractions indicated that the membrane-associated talin was cleaved and reduced to a stable 200-kDa fragment. Conversely, soluble talin remained intact. This 200-kDa fragment was similar to the proteolytic fragment produced from talin by calpain II. Analysis of whole cell extracts and pulse chase experiments indicated that this proteolysis also occurred in vivo, although to a smaller extent. The kinetics of the limited proteolytic cleavage of talin suggested that the 200-kDa fragment was first produced within focal adhesion, and secondarily released into the cytosol.

Animals↗

PubMed1995

Identification of vinculin as a pericentriolar component in mammalian cells.

Previous studies have shown that centrosome position and structure can be influenced by actin filaments, that centrosomes can influence actin organization, and that an actin homologue is associated with centrosomes. Such observations suggest the existence of connections between centrosomes and actin networks. In keeping with such observations, we show that the pericentriolar material, a main component of centrosomes, contains vinculin, a well-known component of cell adhesion plaques and of adherens cell junctions. We find that in various cell types, centrosomes are specifically stained by five different anti-vinculin antibodies. In adherent cell lines, these antibodies also stained adhesion plaques, but in thymocytes, a cell type devoid of adhesive structures, such antibodies stained only centrosomes. Isolated centrosomes also reacted with the anti-vinculin antibodies and immunoelectron microscopy showed apparent localization of vinculin in the pericentriolar material. Immunoblot analysis confirmed the presence of vinculin in purified centrosomal protein preparations. In such protein fractions, anti-vinculin antibodies reacted with a single polypeptide with an apparent molecular weight similar to that of vinculin. Stepwise solubilization of centrosomal structures using urea showed that high urea concentrations were required to solubilize centrosomal vinculin, suggesting tight association of vinculin with the pericentriolar material. The identification of vinculin as a component of centrosomes provides a possible molecular basis for interaction between F-actin and centrosomes.

Animals↗