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To investigate changes in opioid use after supervised exercise therapy and patient education among knee or hip osteoarthritis patients with chronic opioid use.In this cohort study, we linked data from the Good Life with osteoArthritis in Denmark register (GLA:D®; standardised treatment program for osteoarthritis; January 2013 to November 2018) with national health registries. Among 35,549 patients, 1,262 were classified as chronic opioid users based on amount and temporal distribution of dispensed opioids the year before the intervention. We investigated changes in opioid use, measured as mg oral morphine equivalents (OMEQs), from the year before the intervention to the year after using generalized estimating equations.We found a 10% decrease in mg OMEQs from the year before to the year after the intervention (incidence rate ratio [IRR]: 0.90, 95% confidence interval [CI]: 0.86, 0.94). Additional analyses suggested this decrease to be mainly attributable to regulatory actions targeting opioid prescribing during the study period (IRR among patients participating in the intervention before: 0.98 [95% CI: 0.89, 1.07] vs after: 0.83 [0.74, 0.93] regulatory actions). In a random general population sample of matched chronic opioid users, a similar opioid use pattern was observed over time, further supporting the impact of regulatory actions on the opioid use in the study population.Among patients with knee or hip osteoarthritis and chronic opioid use, a standardised treatment program did not change opioid use when regulatory changes in opioid prescribing were taken into account.

Durability requirements for disk brakes in truck application are improved based on the actual customer usage. A second moment reliability index is used to design test codes for the assessment of variable amplitude disk brake fatigue life. The approach is based on the mean estimates of logarithms of equivalent strength of the brake and customer load variables. The index gives possibilities to take all uncertainties in the fatigue life assessment into account, including scatter in material, production, and usage but also systematic errors like model errors in test set up, stress calculations, damage hypothesis, as well as statistical uncertainties.

Background It is becoming increasingly apparent that the extracellular matrix acts as an important regulator of the neural stem niche. Previously we found that neural stem and progenitor cells (NSPCs) derived from the early postnatal subventricular zone of mice adhere to a collagen/hyaluronan hydrogel, whereas NSPCs from the adult and embryonic brain do not. Methods To examine the specific adhesive properties of young stem cells in more detail, NSPCs isolated from embryonic, postnatal day 6 (P6), and adult mouse brains were cultured on collagen I. Results Early postnatal NSPCs formed paxillin-positive focal adhesions on collagen I, and these adhesions could be prevented by an antibody that blocked integrin β1. Furthermore, we found the corresponding integrin alpha subunits α2 and α11 levels to be highest at the postnatal stage. Gene ontology analysis of differentially expressed genes showed higher expression of transcripts involved in vasculature development and morphogenesis in P6 stem cells, compared to adult. Conclusions The ability to interact with the extracellular matrix differs between postnatal and adult NSPCs. General significance Our observations that the specific adhesive properties of early postnatal NSPCs, which are lost in the adult brain, can be ascribed to the integrin subunits expressed by the former furthering our understanding of the developing neurogenic niche. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.

Heparanase functions as a heparan sulfate-degrading enzyme and as a ligand for an unidentified signaling receptor(s). Here, several reactions involved in the activation of the PI3K-AKT pathway by latent heparanase were characterized. Protein suppression using specific siRNAs revealed that heparanase-induced phosphorylation of AKT at Ser-473 was RICTOR-mTOR-dependent, whereas ILK and PAK1/2 were dispensable. p110α was the PI3K catalytic isoform preferred by heparanase for AKT activation and cell proliferation because the p110α inhibitor YM024 blocked these processes. Heparanase-induced AKT phosphorylation was low in mouse embryonic fibroblast cells expressing a RAS interaction-defective p110α compared with wild type cells, indicating that RAS has an important role in the PI3K-AKT activation. The response to heparanase was also inefficient in suspension cultures of several cell lines, suggesting a requirement of integrins in this pathway. Adhesion via either αVβ3 or α5β1 promoted heparanase-induced AKT phosphorylation, and a stronger effect was seen when both integrins were engaged. Simultaneous inhibition of FAK and PYK2 using a chemical inhibitor, or suppression of their expression, inhibited heparanase-induced AKT activation and cell proliferation. Stimulation of cells with heparanase enhanced their resistance against oxidative stress- or growth factor starvation-induced apoptosis. These results demonstrate that there is an intimate cross-talk between the heparanase receptor(s) and integrins during induction of the prosurvival PI3K-AKT pathway by heparanase.

Although a few immunocompetent animal models to study the immune response against human adenoviruses (HAdV) are available, such as Syrian hamsters and cotton rats, HAdV replication is several logs lower compared to human control cells. We have identified a non-transformed mouse epithelial cell line (NMuMG) where HAdV-2 gene expression and progeny formation was as efficient as in the highly permissive human A549 cells. HAdV from species, D and E (HAdV-37 and HAdV-4, respectively) also caused a rapid cytopathic effect in NMuMG cells, while HAdV from species A, B1, B2 and F (HAdV-12, HAdV-3, HAdV-11 and HAdV-41, respectively) failed to do so. NMuMG cells might therefore be useful in virotherapy research and the analysis of antiviral defense mechanisms and the determination of toxicity, biodistribution and specific antitumour activity of oncolytic HAdV vectors.

The piston system accounts for roughly half of the mechanical friction of an internal combustion engine, thus it is important to optimize. Different thermally sprayed cylinder liners were investiga ...

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane-substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5-10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells. We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.

Anticipated emission legislation and reduced fuel consumption are the main driving forces when developing new engines. Optimization of the active surfaces in the piston system is one possible way t ...

Akt, also called PKB, is a serine/threonine kinase that plays a major role in cell survival. It can be activated by several cellular receptors, including integrins and growth factor receptors, in PI3K-dependent manners. In this study, we analyzed the two current models for Akt activation upon beta1 integrin-mediated adhesion: via focal adhesion kinase and via transactivation of the EGF receptor. Distinct differences in the pathways leading to phosphorylation and activation of Akt from stimulated beta1 integrins and EGF receptor were observed, including opposing sensitivity to the tyrosine kinase inhibitors PP2 and Gefitinib. Using knockout cells and integrin mutant cells, we show that beta1 integrins can induce phosphorylation of Akt at Ser473 and Thr308 and Akt kinase activity independently of the EGF receptor activity, focal adhesion kinase, and the Src family members. In contrast to stimulation with EGF, beta1 integrin-mediated adhesion did not induce Akt tyrosine phosphorylation. Moreover, tyrosine phosphorylation of Akt was found not to be required for its catalytic activity. The results identify a previously unrecognized mechanism by which beta1 integrins activate the PI3K/Akt pathway.

Project organizations are often used as tools for accomplishing change in other organizations, which in most cases are their parent organizations. The relationship between these organizations is, consequently, crucial for the implementation of the development activities. This relationship, however, has not been given sufficient attention in contemporary literature on projects. This article has two main purposes: First, to develop a model which may be used for analyzing the relationship between development projects and their parent organizations, particularly the degree of detachment between the involved organizations. The second purpose is to clarify in what way the linkage between the development project and the permanent organization influences the conditions for the implementation of the project’s result. Based on an analysis of case studies conducted in human service organizations, we conclude that implementation depends on how the distinction between the project and permanent organization is originally defined. Project managers thus face a dilemma when organizing development projects; they either organize to accomplish innovation, or to achieve successful implementation.

The medial preoptic nucleus (MPN) regulates sexual behavior which is subject to experience-dependent modifications. Such modifications must depend on functional plasticity in the controlling neural circuits. Thus, MPN synapses are likely candidates for the site of alterations. The present work is a first systematic study of functional synaptic plasticity at glutamatergic synapses in the MPN. Short-term activity-dependent plasticity was investigated using a slice preparation from young male rats. The average efficacy of AMPA/kainate-receptor-mediated synaptic transmission was activity-dependent, showing a peak at a steady stimulation rate of 2 Hz. The variation in efficacy was attributed to mainly presynaptic factors since the average response amplitude was roughly paralleled by the response probability. Upon paired-pulse stimulation, paired-pulse facilitation as well as paired-pulse depression was observed. In some cases, paired-pulse facilitation as well as paired-pulse depression was recorded from an individual neuron depending on the interval between the paired stimuli. On average, paired-pulse facilitation was observed at intervals

In the present study, the functional role of suggested phosphorylation of the conserved threonines in the cytoplasmic domain of integrin subunit beta1 was investigated. Mutants mimicking phosphorylated and unphosphorylated forms of beta1 were expressed in beta1 deficient GD25 cells. T788 in beta1 was identified as a site with major influence on integrin function. The mutation to A788 strongly reduced beta1-dependent cell attachment and exposure of the extracellular 9EG7 epitope, whereas replacement of T789 with alanine did not interfere with the ligand-binding ability. Talin has been shown to mediate integrin activation, but the talin head domain bound equally well to the wild-type beta1 and the mutants indicating that the T788A mutation caused defect integrin activation by another mechanism. The phosphorylation-mimicking mutation T788D was fully active in promoting cell adhesion. GD25 cells expressing beta1T788D accumulated increased number of focal contacts and migrated slowly compared to GD25 beta1 wild-type. An analogous phenotype is seen when focal adhesion kinase activation is abrogated. However, neither the beta1T788D nor the beta1T788A mutation failed to induce tyrosine phosphorylation of focal adhesion kinase. The results suggest that phosphorylation of T788 in integrin beta1 promotes inside-out receptor activation, as well as focal contact accumulation.

Contemporary organization theory reminds us about the impact of the environment on what is going on in organizations and that neither permanent nor temporary organizations can be understood without taking their relations to other organizations into consideration. The aim of this article is to outline a model for analyzing fundamental relationships between projects and important actors in their interaction environment. The model can be used for identifying determinants of uncertainty in the environment and how these influence project structures, processes and outcomes. The model introduce the concept interactional uncertainty which enables us to contrast different ways in which relationships either constrain or enable projects to accomplish their tasks.

The medial preoptic nucleus (MPN) is the major nucleus of the preoptic area (POA), a hypothalamic area involved in the regulation of body-temperature. Injection of capsaicin into this area causes hypothermia in vivo. Capsaicin also causes glutamate release from hypothalamic slices. However, no data are available on the effect of capsaicin on synaptic transmission within the MPN. Here, we have studied the effect of exogenously applied capsaicin on spontaneous synaptic activity in hypothalamic slices of the rat. Whole-cell patch-clamp recordings were made from visually identified neurons located in the MPN. In a subset of the studied neurons, capsaicin enhanced the frequency of spontaneous glutamatergic EPSCs. Remarkably, capsaicin also increased the frequency of GABAergic IPSCs, an effect that was sensitive to removal of extracellular calcium, but insensitive to tetrodotoxin. This suggests an action of capsaicin at presynaptic GABAergic terminals. In contrast to capsaicin, the TRPV4 agonist 4alpha-PDD did not affect GABAergic IPSCs. Our results show that capsaicin directly affects synaptic transmission in the MPN, likely through actions at presynaptic terminals as well as on projecting neurons. Our data add to the growing evidence that capsaicin receptors are not only expressed in primary afferent neurons, but also contribute to synaptic processing in some CNS regions.

The two neurosteroids 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone; AlloP) and pregnenolone sulfate (PregS) affect neuronal GABA(A) receptors differently. While AlloP mainly potentiates the currents through GABA(A) receptors, PregS reduces such currents. The present study aimed at clarifying the interaction of AlloP and PregS at GABA(A) receptors in neurons from the medial preoptic nucleus of male rat. AlloP has previously been shown to dramatically prolong GABA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in these neurons. Here, by recording sIPSCs under voltage-clamp conditions with the perforated-patch technique, it was shown that PregS by itself did not significantly affect the amplitude or time course of such currents. However, PregS, in a concentration-dependent manner, reduced the AlloP-evoked prolongation of sIPSC decay when the two neurosteroids were applied together. In contrast to sIPSC amplitude and time course, sIPSC frequency was significantly reduced by 10 microM PregS alone. Further, although 1.0 microM AlloP alone induced a clear increase in sIPSC frequency, the frequency was not significantly different from control when 1.0 microM AlloP was applied in combination with 10 microM PregS. In addition to the effects on sIPSC parameters, PregS reduced the baseline current evoked by 1.0 microM AlloP in the absence of GABA application or synaptic activity. PregS by itself did not significantly affect the baseline current. The main effects of AlloP and PregS on the sIPSC time course were mimicked by a simplified model with AlloP assumed to reduce the rate of GABA unbinding from the receptor and PregS assumed to increase the rate of desensitization.

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.

The effects of the well-known GABA(A)-receptor blocker bicuculline on voltage-gated K(+) currents were studied in neurons from the medial preoptic nucleus (MPN) of rat. Whole-cell currents were recorded using the perforated-patch technique. Voltage steps from -54 to +6 mV resulted in tetraethylammonium-sensitive K(+) currents of delayed rectifier type. The total K(+) current (at 300 ms), including Ca(2+)-dependent and Ca(2+)-independent components, was reversibly reduced (17 +/- 4%) by 100 microM bicuculline methiodide and (37 +/- 5%) by 100 microM bicuculline as free base. The Ca(2+)-independent fraction (77 +/- 2%) of K(+) current evoked by a voltage step was, however, reduced (54 +/- 6%) only by bicuculline free base, but was not affected by bicuculline methiodide. The half-saturating concentration of bicuculline free base for blocking this purely voltage-gated K(+) current was 113 microM, whereas for blocking a steady Ca(2+)-dependent K(+) current it was 36 microM. The bicuculline-sensitive voltage-gated K(+) current was composed of 4-AP-sensitive and 4-AP-resistant components with different kinetic properties. No component of the purely voltage-gated K(+) current was affected neither by 100 nM alpha-dendrotoxin nor by 100 nM I-dendrotoxin. The possible K(+)-channel subtypes mediating the bicuculline-sensitive current in MPN neurons are discussed.

The effect of perinatal asphyxia on brain development was studied with organotypic cultures from substantia nigra, neostriatum and neocortex. Asphyxia was induced by immersing foetuses-containing uterine horns removed from ready-to-deliver rats into a water bath for 20 min. Following asphyxia, the pups were nursed by a surrogate dam and sacrificed after 3 days to prepare organotypic cultures. Non-asphyxiated caesarean-delivered pups were used as controls. Morphological features were recorded during in vitro development. At day in vitro (DIV) 24, the cultures were treated for histochemistry using fast red for cell nucleus labelling and antibodies against tyrosine hydroxylase for dopaminergic neurons. Compared to controls, cultures from asphyxiated pups revealed a diminished integration quantified during 21 DIV. After immunocytochemistry and camera lucida reconstruction, tyrosine hydroxylase-positive neurons showed a decreased number of neurites from secondary and higher level branching, demonstrating a vulnerability of the dopaminergic systems after perinatal asphyxia.

The endogenous neurosteroid 3alpha-hydroxy-5alpha-pregnane-20-one (allopregnanolone) affects presynaptic nerve terminals and thereby increases the frequency of spontaneous GABA release. The present study aimed at clarifying the mechanisms underlying this presynaptic neurosteroid action, by recording the frequency of spontaneous GABA-mediated inhibitory postsynaptic currents (sIPSCs) in neurons from the medial preoptic nucleus (MPN) of rat. Acutely dissociated neurons with functional adhering nerve terminals were studied by perforated-patch recording under voltage-clamp conditions. It was shown that the sIPSC frequency increased with the external K(+) concentration ([K(+)](o)). Further, the effect of allopregnanolone on the sIPSC frequency was strongly dependent on [K(+)](o). In a [K(+)](o) of 5 mM, 2.0 microM allopregnanolone caused a clear increase in sIPSC frequency. However, the effect declined rapidly with increased [K(+)](o) and at high [K(+)](o) allopregnanolone reduced the sIPSC frequency. The effect of allopregnanolone was also strongly dependent on the external Cl(-) concentration ([Cl(-)](o)). In a reduced [Cl(-)](o) (40 mM, but with a standard [K(+)](o) of 5 mM), the effect on sIPSC frequency was larger than that in the standard [Cl(-)](o) of 146 mM. The dependence of the effect of allopregnanolone on [K(+)](o) and on estimated presynaptic membrane potential was also altered by the reduction in [Cl(-)](o). As in standard [Cl(-)](o), the effect in low [Cl(-)](o) declined when [K(+)](o) was raised, but reversed at a higher [K(+)](o). The GABA(A) receptor agonist muscimol also potentiated the sIPSC frequency. Altogether, the results suggest that allopregnanolone exerts its presynaptic effect by increasing the presynaptic Cl(-) permeability, most likely via GABA(A) receptors.

Polymerization of type I and III collagens is dependent on fibronectin andenhanced by integrins alpha 11beta 1 and alpha 2beta 1.

The importance of the N -methyl- D -aspartate (NMDA) receptor in various painful conditions is well established. The effects of peripheral nerve lesion or joint inflammation, as models of different pain states, on NMDA receptor-mediated currents and NMDA receptor subunit mRNA expression were therefore studied in acutely dissociated neurones from the rat spinal cord dorsal horn. In the neuronal population from control rats, all four NR2 subunits and both NR1 splice variants assayed were detected. A majority of neurones expressed mRNA for more than one NR2 subunit, and some neurones expressed all four NR2 subunits as well as both NR1 splice variants. The NR2B subunit was the most commonly expressed, while the NR2C was the rarest. Following nerve lesion, fewer neurones expressed NR2A compared to the control. The dose–response curve for glutamate-evoked NMDA receptor-mediated currents in the neurones was best described by a three-component fit, suggesting that three functionally distinct NMDA receptor populations are present in the dorsal horn. Minor changes in the dose–response curve after nerve lesion could not be ascribed with certainty to the lesion. Changes in other parameters of NMDA receptor-mediated currents were observed neither after nerve lesion nor after joint inflammation. In summary, the present work demonstrates that single dorsal horn neurones express mRNA for several NMDA receptor subunits. The glutamate dose–response curves indicate that there are three major types of NMDA receptors present in dorsal horn neurones. We also report a reduced expression of NR2A following peripheral nerve lesion.

Recent reports suggest that laminin deposition is controlled by the cell via specific receptors, one of which is dystroglycan. In this study, the involvement of beta1-integrins in this process was investigated by comparing beta1-integrin-deficient cells of different phenotypes with their normal counterparts. Normal embryonic stem (ES) cells and embryoid bodies (EBs) derived from them were found to deposit cell-associated laminin into fibrillar networks, and in the EBs a basement membrane was assembled under the primitive endoderm. beta1-deficient ES cells and their EBs formed only small amounts of dot-like laminin deposits. Skeletal myotubes formed after prolonged differentiation in EBs were found to be surrounded by laminin, nidogen, and perlecan by immunofluorescent staining irrespective of the presence of beta1-integrins on the myotubes. However, at the electron microscope level only very thin sheet-like structures were detected close to the beta1-deficient myotubes, while the wt myotubes formed thick basement membranes. An epithelial cell line, GE11, derived from the beta1-integrin-deficient ES cells was also unable to assemble laminin on the cell surface, while transfection of the cells with the integrin beta1 subunit resulted in formation of a dense laminin network. Taken together, these results suggest that dystroglycan and beta1-integrins can both contribute to the recruitment of laminin to cell surfaces and that integrins are required at a subsequent step in the formation of basement membranes.

Although Staphylococcus aureus is primarily considered an extracellular pathogen, recent evidence suggests that this bacterium can invade a variety of nonprofessional phagocytic cells. Here we investigate the early stages of cellular invasion by S. aureus and determine the bacterial and host components that are required for this process. S. aureus expresses two cell surface-associated fibronectin (FN)-binding proteins (FnbpA and FnbpB) that mediate the interaction of the bacteria with both soluble and solid-phase FN in vitro. Using a mutant of S. aureus that lacks the expression of both Fnbps, we show that the expression of either protein is necessary for efficient uptake by the mouse fibroblast line GD25beta1A. Invasion could be inhibited by soluble recombinant proteins encompassing either the FN-binding D repeat region or the A region (and B repeats) of FnbpA, suggesting that the activities of both regions are important in this process. We demonstrate that FN is also required for invasion of this cell line. In the presence of FN-depleted fetal bovine serum, the invasion level was reduced by approximately 40% compared to in the presence of whole fetal bovine serum. Invasion could be further reduced by the addition of anti-mouse FN antibodies to the assay. Finally, we utilize a mutant mouse fibroblast line, which lacks beta1 integrin expression, to demonstrate that host cell beta1 integrins are necessary for efficient cellular invasion. The level of invasion of the mutant cell line GD25 was reduced by approximately 97% compared to the beta1-expressing complemented cell line GD25beta1A. In addition, invasion of the GD25beta1A cell line could be inhibited by an RGD-containing peptide, further implicating a role for integrins in this process. Based on these observations, we put forward a model of S. aureus invasion in which host FN forms a bridge between the bacterial Fnbps and host cell beta1 integrins, leading to bacterial uptake.

We have expressed the beta1B integrin subunit in beta1-deficient GD25 cells to examine beta1B functions without the interference of endogenous beta1A expression. As previously reported [Retta et al., 1998, Mol. Biol. Cell 9, 715-731], the beta1B integrins did not mediate cell adhesion under normal culture conditions, while the presence of 0.3 mM Mn(2+) allowed beta1B integrins to support adhesion. Mn(2+), as well as the small soluble peptide GRGDS, induced a beta1B conformation, which was recognized by the mAb 9EG7, a marker for active or ligand-bound integrins. beta1B integrins were found to localize to a subset of focal contacts in a ligand-independent manner on fibronectin, but not on vitronectin. However, clustering of beta1B did not induce tyrosine phosphorylation of FAK, p130(Cas), or paxillin, as studied by beta1B-mediated adhesion, to fibronectin in the presence of Mn(2+) or to anti-beta1 antibody in DMEM. Induction of ligand-occupied conformation by the GRGDS peptide during the adhesion to anti-beta1 antibody also failed to trigger FAK phosphorylation. Stimulation of tyrosine phosphorylation on FAK, p130(Cas), and paxillin by adhesion via integrin alphaVbeta3 to fibronectin or vitronectin was not disturbed in GD25-beta1B cells compared to the untransfected GD25 cells, nor were any negative effects of beta1B observed on alphaVbeta3-mediated cell attachment, spreading, and actin organization, or on the cell proliferation rate. These results show that the reported negative effects of beta1B on adhesive events do not apply to alphaVbeta3-dependent interactions and suggest that they may specifically act on beta1 integrins.

In this study we have determined the position of the C-terminal end of the transmembrane domains of human integrin subunits (alpha2, alpha5, beta1, beta2) in microsomal membranes using the glycosylation mapping technique. In contrast to the common view, the transmembrane helices were found to extend roughly to Phe(1129) in alpha2, to Phe(1026) in alpha5, to Ile(757) in beta1, and to His(728) in beta2. The alpha-carbon of the conserved lysine present near the C-terminal end of the transmembrane helix (Lys(1125) in alpha2, Lys(1022) in alpha5, Lys(752) in beta1, and Lys(724) in beta2) is buried in the plasma membrane, and the charged amino group most likely reaches into the polar head-group region of the lipid bilayer. A possible role for the conserved lysine in integrin function is discussed.

Ca2+ spikes, their contribution to firing patterns, and the underlying Ca2+ currents in neurons of the medial preoptic nucleus of rat were investigated by tight-seal whole-cell recordings in a slice preparation. Two different types of spikes were recorded: Low-threshold spikes were generated from membrane potentials-75 mV. High-threshold spikes were recorded when K+ currents were reduced, and were readily evoked from membrane potentials near -40 mV. Both types of spikes were blocked by substitution of Co2+ for Ca2+ in the external medium, but were insensitive to 2.0 microM TTX. Under voltage-clamp conditions, two main types of Ca2+ currents were characterized: low-threshold currents that activated at membrane potentials-60 mV, and high-threshold currents that activated at potentials-30 mV. The low-threshold current and the low-threshold spike were more sensitive to block by external Ni2+ than to block by Cd2+, whereas the high-threshold current and the high-threshold spike were more sensitive to block by external Cd2+ than to block by Ni2+. Significant fractions of the high-threshold currents were blocked by 10 microM nifedipine, 1.0 microM omega-conotoxin GVIA, 50 nM omega-agatoxin IVA and 1.0 microM omega-conotoxin MVIIC, suggesting the presence of L-, N-, P- and Q-type Ca2+ channels. There were also a high-threshold current component insensitive to the above mentioned toxins. It is proposed that the low-threshold current serves as a trigger for short bursts of fast spikes from hyperpolarized levels, whereas the high-threshold current is involved in the Cd2+-sensitive burst firing seen in relatively depolarized neurons.

The adsorption of fibronectin to poly(vinyl chloride) catheters with end-point attached (EPA) heparin and tridodecylmethylammonium chloride-heparinized poly(vinyl chloride) was compared to that of unheparinized poly(vinyl chloride) using antibodies directed against four different domains of the protein. After perfusion of human plasma on the EPA-heparinized surface, the exposure of the N-terminal 29-kD fragment increased during the first 5 h of perfusion. Also, the exposure of the 30-kD gelatin-binding and 65-kD cell-binding fragments increased with time, but at a lower level. On the unheparinized catheter, low levels of antibodies bound to the different domains, and the binding showed little variation during the 5 h of plasma perfusion, indicating that the fibronectin molecule does not change configuration to a significant extent on this surface after the initial adsorption. When the EPA-heparinized surface was preabsorbed with human fibrinogen before incubation with fibronectin, significantly less of the 29-kD (fibrin-binding) domain was exposed, and the 30-kD domain was not exposed. Exposure of the 31- and 65-kD domains increased after preadsorption of fibrinogen to the surface. Since fibronectin has heparin-binding domains, it adsorbs differently to a heparinized versus an unheparinized surface. This will influence subsequent binding of other proteins to the surface, as well as potential binding of microbes. The use of antibodies to defined domains of the fibronectin molecule provides a powerful tool in studies of configurational changes of fibronectin after adsorption to different surfaces.

Preoptic neurons in rat hypothalamic slices were investigated with tight-seal whole-cell recording techniques. The main aim was to investigate the ability to generate graded, stimulus-dependent impulses. In response to rectangular current pulses, all cells generated impulses with an amplitude that to some degree depended on the stimulus strength. Stronger current steps induced impulses of larger amplitude. In 50% of the cells, a systematic variation of impulse amplitude of more than 10 mV (up to 40 mV) was recorded, implying a clear deviation from the 'all-or-nothing' principle. A clear variation in amplitude of spontaneous impulses was also recorded, in the whole-cell mode as well as from intact cells in the cell-attached mode.

The present computational study shows that switch-like membrane properties can be obtained from a model neuron with classical-type Na+ and K+ currents. The membrane potential can adopt two stable steady-state levels and transitions between the two levels may be triggered by positive or negative current stimuli. Thus, the neuron functions in analogy to the flip-flop devices used in computers. In the absence of stimulus input, the neuron may maintain either of the two stable potential levels for infinite durations. Thus, in contrast to plateau-potential generating neurons described earlier, it is suitable for long-term memory. The mechanisms underlying the switch-properties depend entirely on parameter values within the range measured from biological experiments: a relatively large "classical" Na+ current, with respect to K+ and leak currents, is required. It is hypothesized that similar switch-like membrane properties may be found among living neurons in the mammalian brain.

Small cultured hippocampal neurons from rat embryos were studied with the patch-clamp technique. Single-channel currents from outside-out membrane patches were recorded under steady-state potential conditions. The most frequently found channel types were selective to K+ and showed conductances of about 30 and 80 pS in the range −20 to 0 mV. Two basic kinetic patterns were observed for the 80 pS channels. In one type, the fraction of time spent in open state increased with potential, and in the other type it decreased. For both types of 80 pS channel, the distribution of dwell times in the open state was well described by the sum of two exponentials while three exponentials sometimes were required for dwell times in the closed state. The time constants of the fitted exponentials could vary considerably during an experiment.

Three adhesion-related proteins in rat platelets were compared, namely C-CAM, gpIIb/IIIa, and integrin β 1 . These three proteins behaved as distinct components as judged by both biochemical and immunological analyses. GpIIb/IIIa bound to a GRGDSPC-peptide, but neither β 1 -containing integrins nor C-CAM had any affinity either for this peptide or for a large cell-binding fragment of fibronectin. C-CAM and integrin β 1 behaved differently when platelets were labeled with 125 I, solubilized by detergent, and immunoprecipitated. Significant amounts of labeled C-CAM was precipitated when the platelets were first solubilized with detergent and then 125 I-labeled. Almost no labeled C-CAM could be precipitated when intact platelets, that were unactivated or activated by ADP, were labeled. In contrast, labeled integrin β 1 was immunoprecipitated when unactivated platelets were surface-labeled. However, when platelets that were activated by ADP and calcium ions were labeled almost no labeled integrin β 1 could be immunoprecipitated. These data indicate 1) that C-CAM in intact platelets is inaccessible to surface-labeling and 2) that β 1 integrin is less accessible to surface-labeling after platelet activation in the presence of calcium ions.

Attachment of rat hepatocytes to collagen but not to fibronectin substrata was efficiently inhibited by antibodies against the hepatocyte surface. Further analyses of this inhibition suggested that hepatocyte attachment to collagen involves cell surface antigens which are not identical to membrane bound fibronectin or collagen. Rabbit antibodies against rat fibronectin inhibited hepatocyte attachment to rat fibronectin but not to collagen or rabbit fibronectin. After plasmin digestion of fibronectin, peptides were isolated that lacked affinity for collagen but could serve as a substratum for hepatocyte attachment. These results suggested that attachment to fibronectin does not involve membrane bound fibronectin or collagen.

The effect of a cyclic adenosine monophosphate (cAMP) analogue on myelinated axons was studied. The lipophilic dibutyryl cAMP (dbcAMP) was applied to isolated frog axons under voltage clamp conditions. No effect on the ionic currents was observed for either external or internal application of dbcAMP. Nor was any effect of the phosphodiesterase inhibitor theophylline found. These results suggest that cAMP does not play an essential role for nerve impulse conduction in axons. This conclusion deviates from those of other studies.

Extracellular matrix components strongly influence the differentiated properties of isolated rat arterial smooth muscle cells during in vitro cultivation. The attachment and spreading of the cells on a substrate of fibronectin or a 105-kDa cell-binding fragment of fibronectin are accompanied by a structural and functional transformation, referred to as a transition or modulation from a contractile to a synthetic phenotype. Here, the ability of the cell-attachment sequence of fibronectin, Arg-Gly-Asp-Ser (RGDS), to promote this process was studied. The results demonstrate that freshly isolated smooth muscle cells attached to a substrate of the synthetic peptide Gly-Arg-Gly-Asp-Ser-Cys (GRGDSC) in a specific manner and as well as to substrates of fibronectin and the 105-kDa fragment. Subsequent spreading of the cells on the peptide substrate followed the same kinetics and was as extensive as on fibronectin, even if protein synthesis was blocked by treatment of the cultures with cycloheximide. Like fibronectin, the peptide substrate induced formation of actin filament bundles, again without ongoing protein synthesis. Moreover, it was as efficient as fibronectin in supporting the transition of the cells from a contractile to a synthetic phenotype as analyzed by electron microscopy. Antibodies against the β subunit of the fibronectin receptor interfered with the attachment, spreading, and fine structural reorganization of the cells in a similar manner on substrates of fibronectin, the 105-kDa fragment, and GRGDSC. Taken together, the findings indicate that the cell-attachment sequence (RGDS) mimics intact fibronectin in promoting a change in the differentiated properties of arterial smooth muscle cells and does so by interacting with a cell surface receptor for fibronectin.

The spreading behaviour of rat hepatocytes has been investigated. Extensive spreading occurred on substrates made of fibronectin, native collagen fibres, native collagen monomers, and denatured collagen, respectively. An intermediate degree of spreading was observed on substrates made of antibodies directed against the cell surface. The hepatocytes did not show any spreading on substrates made of asialo-ceruloplasmin, concanavalin A or polycationic ferritin, although they readily attached to these substrates. Spreading on denatured collagen and on cell surface antibodies could be inhibited by anti-fibronectin indicating that fibronectin, produced by hepatocytes and immobilized in the substrate, mediated the spreading of cells on these substrates. On the other hand, spreading on native collagen was not inhibited by anti-fibronectin, which suggests that this spreading reaction was not mediated by fibronectin. It was observed that the rate at which hepatocytes spread on collagenous substrates increased with the amount of collagen molecules in the substrate, indicating that spreading of cells is not only triggered by attachment to a proper substrate per se, but depends on the number of adhesion bonds formed by each cell as well.

The spreading of freshly isolated rat arterial smooth muscle cells (SMCs) on a substrate of fibronectin (FN) is associated with marked changes in fine structure and function of the cells, collectively referred to as a modulation from a contractile to a synthetic phenotype. Recent studies have indicated that this process is mediated via an interaction between the minimal cell-attachment sequence of FN (RGDS) and cell surface receptors. Here, we report the isolation of such receptors by sequential chromatography on affinity columns of wheat germ agglutinin (WGA) and a 105-kDa cell-binding fragment of FN (105-kDa fragment). The receptor was composed of two proteins with electrophoretic mobilities in SDS-polyacrylamide gels of 160 and 115 kDa under nonreducing conditions and 150 and 130 kDa under reducing conditions. Immunoprecipitation of surface-labeled cells with a rabbit antiserum against the beta chain of the rat hepatocyte FN receptor similarly yielded two proteins of 160 and 115 kDa. In metabolically labeled cells an additional component of 105 kDa was precipitated, presumably representing a precursor of the 115-kDa protein. Immunocytochemical studies demonstrated that SMCs grown on laminin formed FN fibrils and actin filament bundles in close alignment with cell surface receptors after a few days of culture. In cells seeded on the 105-kDa fragment, the receptors were already arranged in parallel with actin filaments on the first day of culture. Later on, the cells secreted FN and laid down FN fibrils along the receptors on the cell surface and the actin filament bundles in the cytoplasm. Taken together, the findings indicate that arterial SMCs are equipped with FN receptors that belong to the integrin family of proteins and consists of alpha (160-kDa) and beta (115-kDa) subunits. The receptor complexes apparently play an important role in determining the differentiated characteristics of the cells, possibly by mediating a linkage between the extracellular matrix and the cytoskeleton.