Let dietitians, not physicians, write diet prescriptions in hospital settings.
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Adolescent↗
Biomedical subjects
M Pfaff
Publications and source records attributed to M Pfaff.
[Drug use: a life choice or health threat].
The term "enjoyment" is used to denote a pleasurable, utility-creating activity or an act of consumption, which, however, may often be accompanied by subsequent risks, health-care costs or reduction in the quality of life. If viewed from the vantage point of health policy, we may quote several examples of consumer behaviour of this type, ranging first and foremost from the consumption of alcohol and nicotine to the use of "hard" drugs. But certain sports activities or sexual behaviour that may prove damaging to other persons must also be mentioned in this context. All these forms of consumption often lead to direct or indirect consequences to health and thereby result in increased health spending amounting to several thousand million DM. The decline in quality of life resulting from the above-mentioned forms of behaviour could be avoided through health promotion and prevention or, at the very least, through measures of rehabilitation, above all, information, counselling, and education, which represent medium- or long-term measures to that effect. Financial incentives or disincentives, for example, via an increase in co-payment in case of illness caused by risky behaviour, have not been as effective as had been expected by many proponents. Product-specific levies, risk-related payments and other financial incentives and disincentives are generally also not as effective as desired. Health policy measures aimed at influencing behaviour via financial measures are particularly ineffective in case of persons addicted to particular types of consumables, especially to drugs: Information, counselling and education represent non-financial forms of influencing and guiding behaviour with a greater scope for success.
Behavior, Addictive↗
Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses.
The alpha4 integrins are indispensable for embryogenesis, haematopoiesis and immune responses, possibly because alpha4 regulates cellular functions differently from other integrins through its cytoplasmic tail. We used novel mimics of the alpha4 tail to identify molecules that could account for alpha4-specific signalling. Here we report that the alpha4 tail, but not several other alpha-subunit tails, binds tightly to the signalling adaptor paxillin. Paxillin physically associated with alpha4 integrins in Jurkat T cells at high stoichiometry, and joining the alpha4 tail to alphaIIb resulted in a complex of integrin alphaIIbbeta3 with paxillin. This association markedly enhanced the rates of alphaIIbbeta3-dependent phosphorylation of focal adhesion kinase and cell migration. It also reduced cell spreading, focal adhesion and stress fibre formation. A point mutation within the alpha4 tail that disrupts paxillin binding reversed all of these effects. Furthermore, alpha4beta1-dependent adhesion to VCAM-1 led to spreading of mouse embryonic fibroblasts derived from paxillin-null but not from wild-type mice. Thus, the tight association of paxillin with the alpha4 tail leads to distinct biochemical and biological responses to integrin-mediated cell adhesion.
Animals↗
Calpain cleavage of integrin beta cytoplasmic domains.
We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin beta3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin beta subunits, and to map calpain cleavage sites in different integrin beta tails, we treated recombinant cytoplasmic domains of integrin beta1A, beta1D, beta2, beta3 and beta7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin beta cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the beta1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin beta subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs.
Amino Acid Sequence↗
Integrin beta cytoplasmic domains differentially bind to cytoskeletal proteins.
Integrin cytoplasmic domains connect these receptors to the cytoskeleton. Furthermore, integrin-cytoskeletal interactions involve ligand binding (occupancy) to the integrin extracellular domain and clustering of the integrin. To construct mimics of the cytoplasmic face of an occupied and clustered integrin, we fused the cytoplasmic domains of integrin beta subunits to an N-terminal sequence containing four heptad repeat sequences. The heptad repeats form coiled coil dimers in which the cytoplasmic domains are parallel dimerized and held in an appropriate vertical stagger. In these mimics we found 1) that both conformation and protein binding properties are altered by insertion of Gly spacers C-terminal to the heptad repeat sequences; 2) that the cytoskeletal proteins talin and filamin are among the polypeptides that bind to the integrin beta1A tail. Filamin, but not talin binding, is enhanced by the insertion of Gly spacers; 3) binding of both cytoskeletal proteins to beta1A is direct and specific, since it occurs with purified talin and filamin and is inhibited in a point mutant (beta1A(Y788A)) or in splice variants (beta1B, beta1C) known to disrupt cytoskeletal associations of beta1 integrins; 4) that the muscle-specific splice variant, beta1D, binds talin more tightly than beta1A and is therefore predicted to form more stable cytoskeletal associations; and 5) that the beta7 cytoplasmic domain binds filamin better than beta1A. The structural specificity of these associations suggests that these mimics offer a useful approach for the analysis of the interactions and structure of the integrin cytoplasmic face.
Amino Acid Sequence↗
Integrin affinity modulation.
Integrin cell-adhesion receptors mediate interactions between cells and the extracellular matrix. Dynamic regulation of integrin adhesive function is termed 'activation' or 'inside-out' signalling. Activation is key to integrin function in processes as diverse as cell migration, the organization of the extracellular matrix and platelet aggregation. Consequently, there has been an intense effort to elucidate the molecular mechanism of integrin activation. This has resulted in the recent identification of novel cytoplasmic partners for integrins and the emerging characterization of the signal-transduction pathways that regulate integrin 'inside-out' signalling. Here, the authors review the recent developments that have provided us with an increased understanding of the basis of integrin activation.
Animals↗
Aggregation of mononucleated precursors triggers cell surface expression of alphavbeta3 integrin, essential to formation of osteoclast-like multinucleated cells.
Alphavbeta3 is a key integrin mediating adhesion of multinucleated osteoclasts during bone resorption. 1, 25-dihydroxyvitamin D3 upregulates alphavbeta3 integrin expression in mononucleated osteoclast precursors and concomitantly stimulates their differentiation into osteoclasts. This suggests that this integrin could play a major role during osteoclast differentiation. We have developed an in vitro model, in which 1, 25-dihydroxyvitamin D3 sequentially modifies the behavior of macrophages: It first induces rounding up of these cells, then their subsequent aggregation and spreading, which finally leads to cell fusion and the formation of osteoclast-like multinucleated giant cells. We show that, while 1,25-dihydroxyvitamin D3 stimulates the de novo synthesis of alphavbeta3 in macrophages early in this process, its accumulation on the surface is triggered by cell aggregation. A high level of integrin alphavbeta3 cell surface expression correlates with macrophage spreading preceding fusion. This was confirmed by means of novel cell permeable peptides containing the C-terminal sequence of the integrin beta3 tail to specifically block (alphavbeta3 function. Although this peptide has no effect on the aggregation step, it disrupts the spreading of osteoclast precursors and consequently inhibits their fusion. These findings suggest a novel role of the integrin alphavbeta3 in a discrete step of osteoclast differentiation.
Amino Acid Sequence↗
A genetic analysis of integrin function: Glanzmann thrombasthenia in vitro.
Glanzmann thrombasthenia, an inherited bleeding disorder, can be caused by a defect or deficiency in platelet integrin alphaIIb beta3 (GPIIb-IIIa). Studies of thrombasthenia variants have facilitated identification of sites involved in the functions of alphaIIb beta3 and other integrins. Such sites include those that bind ligand and those that participate in the "activation" of alphaIIb beta3 required for high affinity binding of ligands such as fibrinogen or PAC1, a monoclonal antibody. Here we describe the isolation of such variants, created in vitro with Chinese hamster ovary cells that express an activated form of alphaIIb beta3. These cells were exposed to a mutagen, ethyl methane sulfonate, and variants that lost the capacity to bind PAC1 were isolated by fluorescence-activated cell sorting. These variants were grouped into three phenotypic classes. One comprised integrin mutations that disrupt ligand binding function; a second comprised mutations that interfere with the capacity of cells to activate the integrin. Most of these activation-defective mutations were in the integrin cytoplasmic domain, but surprisingly, several were caused by mutations affecting three closely spaced residues in the beta3 extracellular domain. A third class of mutants exhibited a defect in integrin activation not ascribable to changes in the integrin sequence. Thus, these may represent mutated signaling molecules required for integrin activation. This unbiased genetic approach provides new insights into the structural basis of integrin function and may assist in identifying the cellular events that regulate integrin function.
Amino Acid Sequence↗
Suppression of integrin activation: a novel function of a Ras/Raf-initiated MAP kinase pathway.
Rapid modulation of ligand binding affinity ("activation") is a central property of the integrin cell adhesion receptors. Using a screen for suppressors of integrin activation, we identified the small GTP-binding protein, H-Ras, and its effector kinase, Raf-1, as negative regulators of integrin activation. H-Ras inhibited the activation of integrins with three distinct alpha and beta subunit cytoplasmic domains. Suppression was not associated with integrin phosphorylation and was independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlated with activation of the ERK MAP kinase pathway. Thus, regulation of integrin affinity state is a novel, transcription-independent function of a Ras-linked MAP kinase pathway that may mediate a negative feedback loop in integrin function.
Animals↗
Cell adhesion and integrin binding to recombinant human fibrillin-1.
Fibrillin-1 is a major constituent of tissue microfibrils that occur in most connective tissues, either in close association with or independent of elastin. To test possible cell-adhesive functions of this protein, we used recombinant human fibrillin-1 polypeptides produced in a mammalian expression system in cell attachment and solid-phase integrin binding assays. Fibrillin-1 polypeptides containing the single RGD sequence located in the fourth 8-cysteine domain, mediated distinct cell adhesion of a variety of cell lines and bound to purified integrin alphaVbeta3. Integrins alphaIIbbeta3, alpha5beta1, alpha2beta1 and alpha1beta1 did not interact with any of the recombinant fibrillin-1 peptides. Our results indicate a novel role for fibrillin-1 in cellular interactions mediated via an RGD motif that is appropriately exposed for recognition by integrin alphaVbeta3.
Amino Acid Sequence↗
Beta 1 integrin-dependent and -independent polymerization of fibronectin.
The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.
Animals↗
Integrin-binding and cell-adhesion studies of fibulins reveal a particular affinity for alpha IIb beta 3.
The extracellular matrix proteins fibulin-1 (variants C and D) and fibulin-2 occur in basement membranes and in vessel walls and are thus potential candidates for cellular interactions. Recombinant forms of these proteins were obtained from stably transfected kidney cell clones and examined for cell-adhesion activity and binding to five different purified integrins. The two variants of mouse fibulin-1 were inactive in all these assays. Mouse fibulin-2, however, bound to alpha IIb beta 3 integrin almost as strongly as fibrinogen, while a lower activity was found for alpha V beta 3 and almost none for alpha 5 beta 1 integrin. Synthetic SVPRGDLDG peptide, corresponding to the single RGD site of mouse fibulin-2, was a strong antagonist of alpha IIb beta 3 integrin binding. Its affinity for alpha V beta 3 and alpha 5 beta 1 integrins was, however, 10- to 50-fold lower compared to GRGDS. Mouse fibulin-2 also promoted adhesion of thrombin-stimulated platelets and of some established cell lines which could be inhibited by RGD peptides. Human fibulin-2, in which the RGD sequence is changed to RSS, bound less strongly to alpha IIb beta 3 integrin and showed no cell-adhesion activity. Together these data suggest a potential role in hemostatic control for mouse fibulin-2 and possibly also for human fibulin-2.
Amino Acid Sequence↗
Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.
A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.
Animals↗
Binding of purified collagen receptors (alpha 1 beta 1, alpha 2 beta 1) and RGD-dependent integrins to laminins and laminin fragments.
Integrins alpha 1 beta 1 and alpha 2 beta 1 when purified by collagen affinity chromatography, showed distinct binding to mouse tumor laminin-1, which has the chain composition alpha 1 beta 1 gamma 1. The binding was, however, about 10-fold lower than to collagen IV. Only little (alpha 1 beta 1) or no binding (alpha 2 beta 1) was observed to two different laminin isoforms (alpha 2 beta 1 gamma 1, alpha 2 beta 2 gamma 1) from human placenta. Binding to laminin-1 was abolished by EDTA and could be specifically inhibited by antibodies to the respective integrin alpha subunit. These antibodies also inhibited cell adhesion to collagens. The binding of soluble integrins was weaker than that of immobilized integrins but could be enhanced by an activating anti(beta 1 integrin). No enhancement was observed for immobilized integrins. Studies with laminin-1 fragments demonstrated lack of binding to the major cell-adhesive fragment E8 from the long arm, fragments E3 and E4, involved in heparin-binding and self-assembly, respectively, and fragment P1, corresponding to the inner segments of the short arms. A larger short-arm fragment (E1XNd), which lacks the N-terminal beta 1 chain domains V and VI, was as active as laminin. Together, these results, suggested the localization of the binding sites for alpha 1 beta 1 and alpha 2 beta 1 to the N-terminal region of the laminin alpha 1 chain. Fragment P1 but not intact laminin-1 bound to alpha V beta 3 integrin in an EDTA-sensitive and RGD-sensitive manner, underscoring previous data on the cryptic nature of the RGD site in laminin-1. Further analyses by surface plasmon resonance assays demonstrated a KD = 50 nM for alpha 2 beta 1/laminin-1 binding and a KD = 450 nM for alpha V beta 3/fragment P1 binding and confirmed the anti-beta 1-mediated increase in affinity for alpha 2 beta 1.
Amino Acid Sequence↗
Selective recognition of cyclic RGD peptides of NMR defined conformation by alpha IIb beta 3, alpha V beta 3, and alpha 5 beta 1 integrins.
The binding of purified fibrinogen receptor alpha IIb beta 3, vitronectin receptor alpha V beta 3, and fibronectin receptor alpha 5 beta 1 to their corresponding ligands in solid-phase binding assays was used to examine the inhibitory activity of various linear and cyclic penta- and hexapeptides of different conformation containing RGD or RAD sequences. Cyclic peptides with different defined backbone conformations were designed by introducing a single D-amino acid or a proline at different positions in the ring. The data were calibrated for alpha IIb beta 3 integrin incorporated into a planar lipid bilayer by a physical method (total internal reflection fluorescence microscopy) which yielded KD = 1.7 microM for a linear RGD peptide and KD = 0.03 microM for fibrinogen. With this integrin, three cyclic hexapeptides ([GRGDFL], [ARGDFV], [GRGDFV]) were 2-4-fold more inhibitory than the linear GRGDS peptide in solid-phase assays and showed similar inhibition as the fibrinogen ligand. Six peptides had the same or a 2-fold lower activity as the linear reference peptide, and three peptides were up to 7-fold less active. Replacement of Arg or Asp by their stereoisomers or Gly by Ala resulted in a 100-1000-fold reduction in activity. With the two other integrins, a single cyclic pentapeptide [RGDFV] was 10-fold more active (alpha V beta 3) or equal in activity (alpha 5 beta 1) to linear GRGDS, while all of the other cyclic peptides were moderately or distinctly less active. Changes in the RGD sequence caused a less dramatic reduction in binding strength for alpha V beta 3 and alpha 5 beta 1 than for alpha II beta 3. Inhibitory activity was compared with the distance between the C beta atoms of Arg and Asp residues as determined by NMR and indicated that the optimum distance is in the range of 0.75-0.85 nm for alpha IIb beta 3 and at or below 0.67 nm for alpha V beta 3 and alpha 5 beta 1. This indicates that alpha IIb beta 3 less sensitive to variations in the RGD backbone structure and can accommodate a larger distance than the integrins alpha V beta 3 and alpha 5 beta 1.
Amino Acid Sequence↗
IgG monoclonal antibodies that inhibit osteoinductivity of human bone matrix-derived proteins (hBMP/NCP).
Monoclonal hBMP/NCP (human bone morphogenetic protein and associated noncollagenous proteins) antibodies of the IgG class were produced. In vitro, 12 of 19 hBMP/NCP antibodies showed functional inhibition of hBMP/NCP-induced chondroneogenesis in a neonatal muscle tissue assay. Inducing factors were characterized by their inhibiting antibodies with immunoblotting. Several peptide factors seem to be involved in the cascade of induced chondro- and osteogenesis.
Animals↗
Comparison of disintegrins with limited variation in the RGD loop in their binding to purified integrins alpha IIb beta 3, alpha V beta 3 and alpha 5 beta 1 and in cell adhesion inhibition.
The inhibitory capacities of six different disintegrins and one related neurotoxin analogue for the binding of RGD-dependent integrins to either fibrinogen, vitronectin or fibronectin were compared in solid phase assays. Echistatin and flavoridin were the most active inhibitors for alpha V beta 3 and alpha 5 beta 1 integrins and moderately exceeded the activity of the natural protein ligands. The same disintegrins together with eristostatin, bitistatin and barbourin were also very potent inhibitors of fibrinogen binding to alpha IIb beta 3 integrin. For all three integrins, albolabrin showed the lowest affinity, but it still clearly exceeded that of synthetic GRGDS. However, assay conditions may determine these relative affinities, as shown for the alpha IIb beta 3 and alpha V beta 3 integrins when used either in immobilized or soluble form. For alpha IIb beta 3, however, a close correlation was found between KD values determined in platelet binding assays and the concentrations required for half maximal inhibition of three disintegrins. The inhibiting capacity of disintegrins in assays with purified integrins also correlated reasonably well with their inhibition of cell attachment to RGD-dependent protein substrates. However, sequence differences in the RGD loops of the various disintegrins may not fully account for the 20-100-fold difference in their binding capacities. This was particularly evident for echistatin and albolabrin, which differ in this region only by two conservative substitutions but have considerably different inhibitory activities. More remote regions of the disintegrins and alignment of disulfide bridges are therefore likely to contribute to their affinity and selectivity.
Amino Acid Sequence↗
German reform a model for U.S. A reply to Thomas Weil.
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Germany↗