Communities of practice and organizational performance

It identifies four specific performance outcomes associated with the communities of practice studied and links these outcomes to the basic dimensions of social capital.

Temporal clustering with spiking neurons and dynamic synapses: towards technological applications.

We apply spiking neurons with dynamic synapses to detect temporal patterns in a multi-dimensional signal. We use a network of integrate-and-fire neurons, fully connected via dynamic synapses, each of which is given by a biologically plausible dynamical model based on the exact pre- and post-synaptic spike timing. Dependent on their adaptable configuration (learning) the synapses automatically implement specific delays. Hence, each output neuron with its set of incoming synapses works as a detector for a specific temporal pattern. The whole network functions as a temporal clustering mechanism with one output per input cluster. The classification capability is demonstrated by illustrative examples including patterns from Poisson processes and the analysis of speech data.

Transendothelial migration of 27E10+ human monocytes.

The myeloid-related proteins MRP8 (S100A8) and MRP14 (S100A9), two members of the S100 family of calcium-binding proteins, are co-expressed and form a cell-surface and cytoskeleton-associated heterodimer upon calcium mobilization which is recognized by the mAb 27E10. The heterodimer is abundantly expressed in the cytoplasm of granulocytes and a subpopulation of blood monocytes. Previously, we and others demonstrated endothelium-associated MRP8/14 in inflamed tissues in the vicinity of transmigrating leukocytes, suggesting a function of the proteins in this process. Here, we demonstrate that 27E10(+) cells represent a fast-migrating monocyte subpopulation which preferentially utilizes an ICAM-1-dependent mechanism. The following observations imply a function of MRP8/14 in the transmigration process: (i) higher secretion of MRP8/14 from 27E10(+) monocytes compared to 27E10(-) monocytes after interaction with activated endothelium, (ii) higher expression of CD11b on 27E10(+) compared to 27E10(-) monocytes, (iii) up-regulation of CD11b on 27E10(-) monocytes in the presence of MRP14 or MRP8/14 heterodimers but not MRP8 and (iv) active participation of MRP14 but not of MRP8 in transmigration as shown by blocking with respective antibodies. We show that the interaction of 27E10(+) monocytes with activated endothelium leads to MRP8/14 release which may account for the high MRP8/14 concentrations in body fluids of patients with acute or chronic inflammatory diseases. Released MRP8/14 may serve a function by enhancing CD11b expression and/or affinity in human monocytes and by participating in the transendothelial migration mechanism. Thus, MRP8/14 substantially contributes to the recruitment of monocytes to an inflammatory site.

Nuclear hourglass technique: an approach that detects electrically open nuclear pores in Xenopus laevis oocyte.

Nuclear pore complexes (NPCs) mediate both active transport and passive diffusion across the nuclear envelope (NE). Determination of NE electrical conductance, however, has been confounded by the lack of an appropriate technical approach. The nuclear patch clamp technique is restricted to preparations with electrically closed NPCs, and microelectrode techniques fail to resolve the extremely low input resistance of large oocyte nuclei. To address the problem, we have developed an approach for measuring the NE electrical conductance of Xenopus laevis oocyte nuclei. The method uses a tapered glass tube, which narrows in its middle part to 2/3 of the diameter of the nucleus. The isolated nucleus is sucked into the narrow part of the capillary by gentle fluid movement, while the resulting change in electrical resistance is monitored. NE electrical conductance was unexpectedly large (7.9 +/- 0.34 S/cm(2)). Evaluation of NPC density by atomic force microscopy showed that this conductance corresponded to 3.7 x 10(6) NPCs. In contrast to earlier conclusions drawn from nuclear patch clamp experiments, NPCs were in an electrically "open" state with a mean single NPC electrical conductance of 1.7 +/- 0.07 nS. Enabling or blocking of active NPC transport (accomplished by the addition of cytosolic extracts or gp62-directed antibodies) revealed this large NPC conductance to be independent of the activation state of the transport machinery located in the center of NPCs. We conclude that peripheral channels, which are presumed to reside in the NPC subunits, establish a high ionic permeability that is virtually independent of the active protein transport mechanism.

Endothelial protease-activated receptor-2 induces tissue factor expression and von Willebrand factor release.

A second protease-activated receptor (PAR-2) that could be activated by trypsin or more physiologically by mast cell tryptase has been recently cloned. Both the structure and activation mechanism of PAR-2 was similar to the functional thrombin receptor (PAR-1). Although many effects of the coagulation protease thrombin on the vascular endothelium could be attributed to PAR-1 activation, very little is known about the physiological and pathophysiological role of PAR-2. We investigated whether stimulation of PAR-2 on endothelial cells induced two cellular responses that play a central role in primary and secondary haemostasis: the release of high molecular weight von Willebrand factor (hmw-VWF) from Weibel-Palade bodies and the de novo synthesis of tissue factor (TF) mRNA and protein. Human umbilical vein endothelial cells (HUVEC) were incubated with agonists for PAR-2 at 37 degrees C. Both trypsin and SLIGKV increased TF mRNA and activity and induced the release of hmw-VWF due to elevated levels of cytosolic Ca2+. Trypsin (10 nm) induced a 6-fold increase of TF mRNA and reduced time until fibrin clot formation to 37%, indicating trebling of the cell surface located TF activity. Stimulation of HUVEC with the PAR-2 agonist peptide SLIGKV induced a dose-dependent increase of TF mRNA up to 6 times and TF activity up to 3 times. Release of hmw-VWF was achieved both after incubation of HUVEC with trypsin and SLIGKV and was directly depending on intracellular Ca2+ mobilization. To make results comparable to the functional thrombin receptor, homologous experiments were carried out using the PAR-1 agonists thrombin and SFLLRN.

[The Baume du Chevalier de St-Victor otherwise the Baume du Commandeur de Pernes]. / Le Baume du Chevalier de St-Victor alias le Baume du Commandeur de Pernes.

The Baume du Commandeur de Pernes was studied in 1929 and the Commandeur identified by M. Bouvet; the origin of its synonym introduced by N. Lemery (1715), i.e. the Baume du Chevalier de St-Victor had not been elucidated up to now. The author's proposals, based upon genealogical data, are the following: two brothers Chevalier de St-Victor were enrolled in the Order of Malta in 1624 at St Gilles (Languedoc); one of them was able to transmit the Balm's formula to N. Lemery during his stay in Montpellier between 1668 and 1671. The geographically very different origins of the Commander and the Knight should prove perhaps that a basic pharmaceutical formulary common at least to all the French Commanderies was existing at that time. In the other hand, the fluctuations of the formula since the XVIIth century have been reported.

Brefeldin A inhibits thrombin receptor regeneration in human endothelial cells.

Endothelial cells, once stimulated with thrombin, are resistant to subsequent stimulation. After a recovery period of about 60 min the cells are sensitized again for activation by thrombin. The resensitization is independent of the receptor de novo synthesis. Therefore an intracellular pool of thrombin receptors that is possibly co-localized with the Golgi apparatus has been assumed. Brefeldin A (BFA) has been used extensively to investigate the intracellular sorting of proteins because of its dramatic alteration of the structural and functional organization of the Golgi apparatus. Accordingly we have examined the effects of BFA on the regeneration of the thrombin receptor response in human umbilical vein and artery cells. The von Willebrand factor (VWF) release from Weibel-Palade vesicles and the intracellular calcium mobilization were used as physiological parameters of thrombin receptor activation. The addition of BFA (2 microg/ml) to endothelial cells or the reduction of the incubation temperature from 37 degrees C to 16 degrees C blocked the receptor response regeneration almost completely.

[The meetings of the Société de Pharmacie de Paris during troubled periods (1803-1946)]. / Les réunions de la Société de Pharmacie de Paris pendant les périodes troublées (1803-1946).

With newly found archives and other already usable documents, it was possible to study the repercussions of several troubled periods in France on the meetings of the Société de Pharmacie de Paris (1803-1946). It seems that only three meetings were cancelled for 143 years. As a matter of fact, the number of attending members decreased widely in the troubled times. Allusions to contemporary events are not scarce in the minutes and have been reported.

[About two unpublished autographs of Louis Pasteur belonging to the Academie national de pharmacie]. / Sur deux autographes inédits de Louis Pasteur appartenant à l'Académie nationale de pharmacie.

Two unpublished autographs of Louis Pasteur, dated respectively november 26th 1853 and december 22nd 1853 in Strasbourg, were found in unclassified archives of the Academie nationale de pharmacie - Paris. They are described in the paper. They are related to the prize of F 1500 given to him by the Societe de pharmacie, the previous name of the Academy. This award concerned his famous studies on the racemic or paratartric acid.

Trypsin induced von Willebrand factor release from human endothelial cells in mediated by PAR-2 activation.

Nystedt and co-workers cloned in 1994 a second protease activatable receptor (PAR-2) that could be activated by trypsin but not by thrombin (1). In this study, we investigated whether trypsin induced stimulation of endothelial cells is linked to PAR-2 activation. We have found by mRNA analysis that endothelial cells of venous and arterial origin express both protease activatable receptors. The functional thrombin receptor and the protease activated receptor-2 (PAR-2) mediate apparently the same effects in human vascular endothelial cells. Both, the activation of the thrombin receptor with thrombin or SFLLRN and the activation of the PAR-2 with trypsin or SLIGRL induced intracellular calcium mobilisation and a subsequent release of von Willebrand factor (vWf) from Weibel-Palade bodies. As a consequence, it can be concluded that endothelial cells have two different receptors mediating the same cellular responses after activation.

Effect of polyvinyl chloride plastic on the growth and physiology of human umbilical vein endothelial cells.

In this work, human umbilical vein endothelial cells were cultured on common polystyrene cell culture plates, referred to as control plates, as well as on soft polyvinyl chloride plastics (PVC). Growth of human umbilical endothelial cells (HUVEC) on PVC coated with gelatin, collagen A and heparin plasma was significantly less than that on the control plates coated with the same substance or fibronectin. Cells cultured on PVC produced up to four times as much tissue plasminogen activator than control cells. With reference to plasminogen activator inhibitor 1 (PAI-1), more PAI-1 was released from cells grown on PVC than from those on the control plates coated with gelatin and collagen A. After endotoxin stimulation, the PAI-1 release of HUVEC cultured on PVC was significantly higher than that of control cells with the exception of cells grown on the fibronectin-coated PVC that showed no difference. It is concluded that the type of plastic and coat used to culture HUVEC play a definite role in their growth and function.

Regulation of the thrombin receptor response in human endothelial cells.

At present only little information is available about the regulation of the thrombin receptor activity in human endothelial cells. The study presented was performed to clarify the problem of thrombin receptor regulation in human endothelial cells. Endothelial cells were isolated from the vein or artery of human umbilical cords. These cells were stimulated with thrombin or with the thrombin receptor activation peptides (TRAP) SFLLRN or SFLLRNPNDKYEPF and subsequently the von Willebrand factor (vWf) release was measured as a physiologically significant response regarding the thrombin receptor activation. Shortly after the first activation by thrombin or SFLLRN, the cells were desensitised to a second stimulation. After a recovery phase of 10 min, merely 35% of the receptor response was obtained by subsequent stimulation. Expanding the recovery time to 90 min resulted in a vWf release of nearly 100% of the amount measured after the first stimulation. The resensitisation rate was similar for thrombin and SFLLRN stimulated cells. Adding RNA synthesis inhibitors or protein synthesis inhibitors had no effect on the recovery of the receptor response. Therefore, a de novo synthesis of receptor protein must be excluded as a means of resensitising endothelial cells to thrombin. It was shown that the dephosphorylation of tyrosine kinase inactivated receptors is also not responsible for receptor regeneration. These results prove that endothelial cells are capable of developing full thrombin receptor activity after a relatively short recovery phase of only 60-90 min as compared to the recovery phase of 16-24 h in megacaryoblastic cell lines. Desensitisation is similar in thrombin and TRAP stimulated cells. We assumed a transport mechanism for intracellular stored vesicles for receptor resensitisation; a co-localisation with vWf in the Weibel-Palade bodies is improbable.

The tethered ligand receptor is the responsible receptor for the thrombin induced release of von Willebrand factor from endothelial cells (HUVEC).

The present study was undertaken to define clearly the receptor, which is responsible for the thrombin induced vWf release from HUVEC. Vu et al. reported that cleavage of the platelet thrombin receptor by thrombin resulted in a new N-terminus (SFLLRN...) which acts as tethered ligand (4). The free peptide activates platelets and induces rises in both cytosolic free Ca2+ and PGI2 production in HUVEC (10). HUVEC were incubated with thrombin, SFLLRN or other relevant substances. After incubation, the intracellular vWf content was compared with the vWf concentration in the supernatant. The intracellular vWf concentration was measured by microscope fluorometry and the concentrations in the supernatant with an ELISA. The thrombin stimulated cells showed 53% vWf antigen compared with control cells (100%). This result was well correlated with the 2.4 fold higher vWf concentrations measured in the supernatant of thrombin stimulated cells than in control cells. Also the addition of SFLLRN (1-60 microM) or trypsin (1-50 nM) increased vWf release from HUVEC in a dose dependent manner. These results indicate that thrombin induced vWf release from HUVEC is mediated through the activation of the tethered ligand receptor.

The influence of heparin and protamine sulfate on platelet ADP and platelet factor 4 release and the expression of glycoprotein IIb/IIIa.

We present results concerning the pathogenic mechanism of heparin-induced thrombocytopenia type I. An ELISA was developed for directly measuring the expression of glycoprotein IIb/IIIa (GPIIb/IIIa) on platelets in the presence and absence of ADP and under the influence of various heparins. In addition, the release of ADP and platelet factor 4 (PF4) was measured in platelet-rich plasma. Heparin also induced the expression of GPIIb/IIIa without prior stimulation with ADP. On the other hand we could demonstrate that the addition of heparins and protamine sulfate to platelets resulted in a significant release of intracellularly stored ADP and PF4. These results suggested that heparin(oid)s modulate the expression of GPIIb/IIIa with ADP as a mediator, and that protamine sulfate is contraindicated as an antidote in heparin-induced thrombocytopenia.

The inhibition of thrombin and chymotrypsin by heparin-cofactor II.

Heparin cofactor II (HC II) is known as a bifunctional inhibitor inactivating trypsin- and chymotrypsin type proteases. Its inhibitory activity increases in the presence of heparin, dermatan sulfate and chondroitin E. In the present study the inhibitory activity of HC II was investigated as function of various dermatan sulfate fractions and its stability was tested against oxidation reagents similar to thus secreted by activated leucocytes. High affinity dermatan sulfate (DS) increased the antithrombin inhibition activity of HC II about 1000-fold in contrast to about 100-fold in the case of low affinity DS. Oxidation of HC II carbohydrate side chains with sodium periodate showed less inactivation effects than oxidation by chloramine T or ammonium peroxodisulfate.

Binding of urapidil to human serum albumin: dependency on free fatty acid concentration.

The interaction between free fatty acids and urapidil binding to human serum albumin (HSA) was investigated, whereby the binding properties at the coumarine binding site were influenced by the HSA fatty acid content. As has been reported for the coumarine derivative warfarin, the highest affinity of urapidil to HSA was found at the albumin fatty acid molar ratio of 1:3. Increasing the fatty acid concentration from 0 to 3 moles per mol human serum albumin caused an increase from 50% to 95% of bound urapidil. The affinity constant increased from 2.2 x 10(3) M-1 to 2.0 x 10(4) M-1 but the number of binding sites remained constant (n = 1.2). Therefore, one urapidil binding site per HSA molecule may be assumed.

Influence of two guar preparations on glucose and insulin levels during a glucose tolerance test in healthy volunteers.

Dietary fiber deficiency may be alleviated with an increase in fiber-rich foods or fiber in the form of guar. The efficacy of a solid guar preparation and a liquid form on glucose and insulin levels during a glucose tolerance test in 12 healthy volunteers was examined. The first half of the study tested the efficacy of a new guar preparation (GU-052, Steigerwald, Darmstadt, Germany). Six volunteers ingested 75 glucose and 12 g guar mixed in 450 ml water. The other 6 received the same drink without guar. Blood was withdrawn for the determination of glucose and insulin before ingesting the glucose solution and after 15, 30, 45, 60, 90, 120, 180, 210 and 240 min. One week later the groups were crossed over. The second half of the study tested the efficacy of GU-052 against a solid preparation consisting of 5 g guar and 1.13 g additive (Glucotard, Boehringer, Mannheim, Germany). Volunteers receiving GU-052 with the glucose drink showed no increase in plasma glucose. Volunteers without GU-052 reached a maximal plasma glucose value of 6.66 +/- 1.30 mmol/l 30 min after glucose ingestion from an initial value of 5.28 +/- 0.40 mmol/l. A second smaller peak was observed 120 min after glucose ingestion. A 2.5-fold increase from 16.17 +/- 4.30 to 40.10 +/- 19.00 microIU/ml in insulin concentration occurred in the volunteers ingesting GU-052, whereas a 6-fold increase from 19.33 +/- 12.20 to 114 +/- 48.8 microIU/ml insulin occurred in the volunteers without GU-052. A second peak in insulin at 120 min which occurred in the volunteers without GU-052 was not apparent when the volunteers ingested GU-052. The comparison of GU-052 and Glucotard showed that GU-052 maintained the glucose level, whereas Glucotard allowed the plasma glucose to reach 6.67 +/- 0.98 microIU/l. The increase in insulin was not as great with GU-052 as with Glucotard. This study shows that guar suppresses glucose and insulin levels during a glucose tolerance test. The guar form influences the efficacy.

Effects of urapidil on 5-hydroxytryptamine induced platelet aggregation and on 14C-5-hydroxytryptamine uptake in platelets.

Urapidil inhibits the 5HT induced human platelet aggregation and the 5HT uptake by platelets in vitro and ex vivo. The aggregation was inhibited with a KI-value of 8.8 microM and the 5HT uptake in a noncompetitive way with KI1 = 15.3 microM and K12 = 10.6 microM. In vivo, the 5HT induced platelet aggregation was increased 4 h after oral administration of 60 mg urapidil. Subsequently, the aggregation decreased and reached a minimum at various times in the different volunteers. Eight h after oral administration of 60 mg urapidil, the 5HT uptake velocity was reduced approximately 40%.

Urapidil inhibits 5-hydroxytryptamine induced platelet aggregation and 14C-5-hydroxytryptamine uptake in platelets.

The effect of urapidil, an a1-antagonist with additional antihypertensive action via central 5HT1A agonism, was determined on platelet aggregation induced by 5HT and adrenaline. The 5HT uptake in human platelets was determined as well. Urapidil inhibited both the 5HT and adrenaline-induced human platelet aggregation and the 5HT uptake by platelets in vitro. The 5HT-induced aggregation was inhibited with a K1 value of 8.8 microM, the adrenaline-induced aggregation with a K1 value of 21.6 microM, and the 5HT uptake non-competitively with a K11 = 11.5 microM and a K12 = 13 microM.