Cooperativity between integrin activation and mechanical stress leads to integrin clustering.

Integrins are transmembrane receptors involved in crucial cellular biological functions such as migration, adhesion, and spreading. Upon the modulation of integrin affinity toward their extracellular ligands by cytoplasmic proteins (inside-out signaling) these receptors bind to their ligands and cluster into nascent adhesions. This clustering results in the increase in the mechanical linkage among the cell and substratum, cytoskeleton rearrangements, and further outside-in signaling. Based on experimental observations of the distribution of focal adhesions in cells attached to micropatterned surfaces, we introduce a physical model relying on experimental numerical constants determined in the literature. In this model, allosteric integrin activation works in synergy with the stress build by adhesion and the membrane rigidity to allow the clustering to nascent adhesions independently of actin but dependent on the integrin diffusion onto adhesive surfaces. The initial clustering could provide a template to the mature adhesive structures. Predictions of our model for the organization of focal adhesions are discussed in comparison with experiments using adhesive protein microarrays.

Excitable waves at the margin of the contact area between a cell and a substrate.

In this paper, we study a new physical mechanism to generate an activator field which signals the extreme margin of the contact area between an adherent cell and the substrate. This mechanism is based on the coupling between the adhesive bridges connecting the substrate to the cytoskeleton and a cytosolic activator. Once activated by adhesion on the adhesive bridges, this activator is free to diffuse on the membrane. We propose that this activator is part of the mecano-transduction pathway which links adhesion to actin polymerization and, thus, to cellular motility. The consequences of our model are as follows: (a) the activator is localized at the rim of the contact area, (b) the adhesion is reinforced at the margin of the contact area between the cell and the substrate, (c) excitable waves of the activator can propagate along the adhesion rim.

Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor.

An N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) has recently been purified on the basis of its ability to restore transport to NEM-inactivated Golgi membranes in a cell-free transport system. NSF is a peripheral membrane protein required for the fusion of transport vesicles. We now report the existence of two novel components that together bind NSF to Golgi membranes in a saturable manner. These components were detected by examining the requirements for reassociation of purified NSF with Golgi membranes in vitro. One component is an integral membrane receptor that is heat sensitive, but resistant to Na2CO3 extraction and to all proteases tested. The second component is a cytosolic factor that is sensitive to both proteases and heat. This soluble NSF attachment protein (SNAP) is largely resistant to NEM and is further distinguished from NSF by chromatography. SNAP appears to act stoichiometrically in promoting a high-affinity interaction between NSF and the membrane receptor. Because NSF promotes vesicle fusion, it seems likely that these two new factors that allow NSF to bind to the membrane are also part of the fusion machinery.

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.

Substrate-site interactions in the membrane-bound adenine-nucleotide carrier as disclosed by ADP and ATP analogs.

The binding parameters of a number of ADP or ATP analogs to the adenine nucleotide carrier in mitochondria and inside-out submitochondrial particles have been explored by means of two specific inhibitors, carboxyatractyloside and bongkrekic acid. The nucleotides tested fell into two classes depending on the shape of the binding curve. Curvilinear Scatchard plots were obtained for the binding of ADP, ATP, adenosine 5'-triphospho-gamma-1-(5-sulfonic acid)naphthylamidate [gamma-AmNS)ATP) and adenylyl (beta,gamma)-methylenediphosphate (p[CH2]ppA); on the other hand, rectilinear Scatchard plots were obtained in the case of naphthoyl-ADP (N-ADP) and 8-bromo ADP (8Br-ADP) binding. The total number of binding sites for N-ADP and 8Br-ADP could be extrapolated with good accuracy to 1.3-1.5 nmol/mg protein; this value corresponds to the number of carboxyatractyloside-binding sites in heart mitochondria (Block, M.R., Pougeois, R. and Vignais, P.V. (1980) FEBS Lett. 117, 335-340). On the other hand, because of the curvilinearity of the Scatchard plots for the binding of ADP, ATP, (gamma-AmNS)ATP and p[CH2]ppA, the total number of binding sites for these nucleotides could only be approximated to a value higher than 1 nmol/mg protein, the exact value being probably equal to that found for N-ADP and 8Br-ADP binding, i.e. 1.3-1.5 nmol/mg protein. Curvilinearity of Scatchard plots was discussed in terms of negative interactions between nucleotide-binding sites located on the same face of the adenine nucleotide carrier. A possible relationship between the features of the binding plots and the transportable nature of the nucleotide is discussed. Contrary to the enhancing effect of bongkrekic acid on [14C]ADP uptake observed essentially in nucleotide-depleted heart mitochondria (Klingenberg, M., Appel, M., Babel, W. and Aquila, H. (1983) Eur. J. Biochem. 131, 647-654), binding of bongkrekic acid to nondepleted heart mitochondria was found to partially displace previously bound [14C]ADP. These opposite effects of bongkrekic acid may be explained by assuming that bongkrekic acid is able to abolish negative cooperativity between external (cytosolic) ADP-binding sites.

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.

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.

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.

Medical comorbidity of major depressive disorder in a primary medical practice.

Despite much speculation about the relationship between depression and medical comorbidity in primary care settings, few investigators have examined this issue empirically. Using a two-stage screening procedure, we assessed 618 patients aged 18 to 64 years in an academic general medicine clinic. Forty-one patients (6.6%) suffered from a current episode of major depressive disorder (MDD). We compared this group with a 20% random sample of nondepressed patients. While patients with MDD were younger (mean age, 41.1 vs 47.2 years), they were assessed by the Duke University Severity of Illness Scale as having more severe medical illness. Patients with MDD were more likely to have malignant tumors and "ill-defined conditions" than nondepressed patients. The 18 patients with MDD (44%) who were correctly diagnosed by their physicians had less severe medical illness than those whose depression was clinically undetected. A logistic regression model predicting MDD group membership included female gender, younger age, higher Duke University Severity of Illness Scale score, and more frequent inactive ill-defined diagnoses. These findings are consistent with assertions: (1) patients with MDD have more physical illness than nondepressed patients and/or (2) somatic symptoms and disability caused by MDD add to the burden of physical illness.

Integrin-mediated adhesion as self-sustained waves of enzymatic activation.

Integrin receptors mediate interaction between the cellular actin-cytoskeleton and extracellular matrix. Based on their activation properties, we propose a reaction-diffusion model where the kinetics of the two-state receptors is modulated by their lipidic environment. This environment serves as an activator variable, while a second variable plays the role of a scaffold protein and controls the self-sustained activation of the receptors. Due to receptor diffusion which couples dynamically the activator and the inhibitor, our model connects major classes of reaction diffusion systems for excitable media. Spot and rosette solutions, characterized by receptor clustering into localized static or dynamic structures, are organized into a phase diagram. It is shown that diffusion and kinetics of receptors determines the dynamics and the stability of these structures. We discuss this model as a precursor model for cell signaling in the context of podosomes forming actoadhesive metastructures, and we study how generic signaling defects influence their organization.

Treating depression in primary care practice. An application of decision analysis.

Decision analysis approaches complex treatment issues by considering alternative strategies in an explicit and logical manner, and examining their outcomes in the face of varied assumptions. Significant data gaps impede full application of this framework to the treatment of depressed primary care patients. Nevertheless, decision analysis already can be useful in emphasizing needed clinical information in treating these patients and highlighting future directions for research.

Strategies for evaluating treatments for major depression in primary care patients.

Primary care physicians are being urged to provide patients experiencing a major depression treatments validated with psychiatric patients. The propriety of transferring clinical technologies from one care-giving sector to another is questionable, however, as it has little scientific support. We suggest that clinical trials be initiated so as to expand the available knowledge base. This paper analyzes the methodologic issues involved in pursuing such experimental research and urges that it be conducted despite the possible need for initial design compromises.

Frames and heuristics in doctor-patient discourse.

The medical interview can be viewed as a verbal exchange between two people trying to solve a problem. Research in linguistics and cognitive psychology casts interesting and useful light on such discourse. Using the concepts 'frames' and 'heuristics' from these fields, we viewed, transcribed and analyzed taped sessions between a family practice physician and sixteen patients to examine how those patients understood their illness. To be an effective teacher the physician must discover not only the causes of the patient's medical problem, but also something of the conceptual structure that supports the patient's perceptions and understanding of his problem. This paper presents a method to understand these conceptual structures and how they function.

A taxonomy of difficult physician-patient interactions.

A taxonomy of troublesome physician-patient interactions helps learners classify what goes wrong during an interview and then remedy the problem in order to obtain reliable symptom data. This framework permits an orderly approach to interactions that often become laden with emotion and also emphasizes the use of specific skills to acquire accurate and precise medical information. Like most systems of classification in medicine, this one is a mixed bag of nosology; although based on the needs of learners, it is arbitrary and imperfect and will change as new knowledge is integrated.

Psychiatric disorders in primary care: physician judgements of prevalence and management.

Primary care physicians underdiagnose depression, anxiety, and other psychiatric disorders. Decision analysis suggests that subjective estimates of the probability of a condition and the utility (severity and treatability) of identifying that condition play a role in diagnosis. We asked 108 internists (IM) and family practitioners (FP) to rank 25 conditions on ten-point scales for probability, severity, and treatability. FPs ranked depression significantly higher than IMs did on all three scales, anxiety reactions higher in probability and severity, and both alcoholism and drug dependency higher on the severity scale. Ranks for schizophrenia and personality disorder did not differ between specialties. Thirty-eight physicians completed the scales a second time after six to eight weeks. Test-retest agreement ranged from 47% to 100% for different conditions on different scales. Some common psychiatric disorders tended to have low test-retest agreement in probability and severity. With further refinement, this instrument may contribute to the investigation of psychiatric decision making in primary care.

Ethical problems of recording physician-patient interactions in family practice settings.

Recordings of actual physician-patient interactions are an important tool for family medicine education and research. Their use, however, poses two sets of ethical problems: one dealing with privacy and confidentiality, and another related to limitations upon informed consent in the context of ordinary medical care. Experience with audiotaping and videotaping led to engaging in a "principle-based" method of ethical reasoning in which problems generated by difficult cases were examined in light of both current rules or guidelines and four fundamental ethical principles. Through this approach specific policies were developed for voluntary, informed consent and for protection of privacy, while recognizing that each case must be judged in the light of the physician's obligation to do the best for each patient.