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Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face-scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three-dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre-scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three-dimensional analysis. Here, we explore focused ion beam facilitated serial block face-scanning electron microscopy to study the endothelial cell–specific storage organelles, the Weibel–Palade bodies, during their biogenesis at the Golgi apparatus. Weibel–Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face-scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel–Palade bodies and Golgi stacks which became conceivable in large-scale three-dimensional data. We demonstrate that serial block face-scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.

SummaryWeibel-Palade bodies (WPBs) are rod or cigar-shaped secretory organelles that are formed by the vascular endothelium. They contain a diverse set of proteins that either function in haemostasis, inflammation, or angiogenesis. Biogenesis of the WPB occurs at the Golgi apparatus in a process that is dependent on the main component of the WPB, the haemostatic protein von Willebrand Factor (VWF). During this process the organelle is directed towards the regulated secretion pathway by recruiting the machinery that responds to exocytosis stimulating agonists. Upon maturation in the periphery of the cell the WPB recruits Rab27A which regulates WPB secretion. To date several signaling pathways have been found to stimulate WPB release. These signaling pathways can trigger several secretion modes including single WPB release and multigranular exocytosis. In this review we will give an overview of the WPB lifecycle from biogenesis to secretion and we will discuss several deficiencies that affect the WPB lifecycle.

Weibel-Palade bodies (WPBs) comprise an on-demand storage organelle within vascular endothelial cells. It's major component, the hemostatic protein von Willebrand factor (VWF), is known to assemble into long helical tubules and is hypothesized to drive WPB biogenesis. However, electron micrographs of WPBs at the Golgi apparatus show that these forming WPBs contain very little tubular VWF compared with mature peripheral WPBs, which raises questions on the mechanisms that increase the VWF content and facilitate vesicle growth. Using correlative light and electron microscopy and electron tomography, we investigated WPB biogenesis in time. We reveal that forming WPBs maintain multiple connections to the Golgi apparatus throughout their biogenesis. Also by volume scanning electron microscopy, we confirmed the presence of these connections linking WPBs and the Golgi apparatus. From electron tomograms, we provided evidence that nontubular VWF is added to WPBs, which suggested that tubule formation occurs in the WPB lumen. During this process, the Golgi membrane and clathrin seem to provide a scaffold to align forming VWF tubules. Overall, our data show that multiple connections with the Golgi facilitate content delivery and indicate that the Golgi appears to provide a framework to determine the overall size and dimensions of newly forming WPBs.

Revealing the ultrastructure and function of fluorescently labeled cellular components by correlative light and electron microscopy (CLEM) facilitates the study of structure-function relationships in complex biological processes. Given the diversity of available fluorescent tags, light microscopy is ideal for monitoring dynamic cellular processes, while electron microscopy reveals the morphological context of structures at high resolution. Endothelial cells lining the blood vessel wall contain storage organelles called Weibel-Palade bodies (WPBs), which contain tubules of densely packed helical spirals of the blood coagulation protein Von Willebrand factor (VWF). Exocytosis of WPBs is triggered upon vascular damage and results in the transformation of stored tubular VWF into secreted extracellular VWF. Upon exocytosis, VWF rearranges into long filamentous strings to recruit platelets from the blood. During this secretion process, large intracellular VWF exocytosis structures are formed called secretory pods. Here, we describe a CLEM method used to study the relationship between the secretory pod and secreted VWF where confocal microscopy on whole cells was combined with serial electron tomography on chemically fixed, plastic-embedded sections. We show that the combination of these two well-established microscopy modalities provides a robust and generic CLEM method suitable for the characterization of VWF secretion sites.

Von Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

Summary Background In vascular endothelial cells, high molecular weight multimers of von Willebrand factor (VWF) are folded into tubular structures for storage in Weibel–Palade bodies. On stimulation, VWF is secreted and forms strings to induce primary hemostasis. The structural changes composing the transition of stored tubular VWF into secreted unfurled VWF strings are still unresolved even though they are vital for normal hemostasis. The secretory pod is a novel structure that we previously described in endothelial cells. It is formed on stimulation and has been postulated to function as a VWF release site. In this study, we investigated the actual formation of secretory pods and the subsequent remodeling of VWF into strings. Methods Human umbilical vein endothelial cells were stimulated and studied using various imaging techniques such as live-cell imaging and correlative light and electron microscopy. Results We found by using live-cell imaging that secretory pods are formed through the coalescence of multiple Weibel–Palade bodies without involvement of other large structures. Secreted VWF expelled from secretory pods was found to adopt a globular conformation. We visualized that VWF strings derive from those globular masses of VWF. Flow experiments showed that, on secretion, the globular masses of VWF move to the edge of the cell, where they anchor and generate VWF strings. Conclusion On secretion, VWF adopts a globular conformation that remodels into strings after translocation and anchoring at the edge of the cell. This finding reveals new pathophysiological mechanisms that could be affected in patients with von Willebrand disease.

Regulated exocytosis of Weibel-Palade bodies (WPBs) is a pivotal mechanism via which vascular endothelial cells initiate repair in response to injury and inflammation. Several pathways have been proposed to enable differential release of bioactive molecules from WPBs under different pathophysiologic conditions. Due to the complexity, many aspects of WPB biogenesis and exocytosis are still poorly understood. Herein, we have investigated the regulated exocytosis of the major WPB constituent, von Willebrand Factor (VWF), which upon its release forms strings of up to several millimeters long that capture circulating platelets and thereby initiate the formation of a haemostatic plug. Using correlative, fluorescence, and electron microscopic imaging techniques, we provide evidence that multigranular exocytosis is an important pathway for VWF release in secretagogue-challenged human umbilical vein endothelial cells. A novel membrane-delimited structure (secretory pod) was identified as the site of WPB coalescence and VWF exocytosis. Clathrin-coated profiles present on the secretory pods suggested remodeling via compensatory membrane retrieval. Small, 30- to 40-nm cytoplasmic vesicles (nanovesicles) mediated the fusion of WPBs with secretory pods. Multigranular exocytosis may facilitate VWF string formation by pooling the content of multiple WPBs. In addition, it may provide a novel mechanism for the differential release of WPB cargo.

We carried out an epidemiological study on thr prevalence and annual incidence of myasthenia gravis on tropical islands Curacao and Aruba in the period 1980 1995. Twenty-one patients (seven men and 14 women) were identified. The point prevalence increased from 29 per million in 1980 to about 70 per million in 1990-1995; the annual incidence over the total period was 4.7 per million. The female:male ratio was 2:1; purely ocular cases (2/21) comprised 9.5% and thymomas (4/21), 19%. These data are in accordance with most other epidemiological studies in non-tropical areas. No other studies on myasthenia gravis in tropical areas have been reported. (C) 2000 Elsevier Science B.V. All rights reserved.

Fifty-three patients with migraine, recruited from the Dutch Society of Migraine Patients and a general practice, were investigated regarding pain, moods, thoughts, and functioning during their most recent migraine attack, using a semistructured interview. Salient findings were: the high pain intensity the patients endured before they took analgesic medication, concerns about medication damaging their health, overoptimism regarding the effect of analgesic medication, and the relatively large proportion of patients (43%) who took medication primarily to be able to continue their activities. We recommend that future clinical trials on the effects of medication on migraine should not only include the measurement of pain during the attack, but also emotions, concerns about potential side effects and the ability to continue or resume work. Furthermore, it is important to provide patients with information about the side effects of medication and to apply cognitive-behavioral techniques for improvement of their mood during the attack.

Abstract 1072 Background: Von Willebrand Factor (VWF) is synthesized in endothelial cells and megakaryocytes and is either secreted constitutively into plasma or stored in specific organelles; Weibel-Palade bodies (WPB) in endothelial cells or α -granules in megakaryocytes and platelets. Release of von Willebrand factor from WPB in response to desmopressin (DDAVP), an agonist of WPB exocytosis, is clinically applied to raise VWF plasma levels in patients with von Willebrand disease (VWD). A subset of patients with VWD type 1, a quantitative defect, shows a reduced response to DDAVP. This reduced response suggests the absence of recruitable WPB. The VWF propeptide (D1-D2 domains) together with the D'D3 domains are necessary for the tubular assembly of VWF. The assembly of VWF into tubules drives WPB formation. Some variants in the VWF A1 to A3 domains have been linked to reduced DDAVP responsiveness. This suggests that determinants for WPB formation may reside outside the D1-D2-D'-D3 domains. We hypothesize that a reduced tendency to assemble into VWF tubules underlies the defective DDAVP response in VWD type 1 patients with mutations in the A domains of VWF. Methods: Human Embryonic Kidney (HEK) 293 cells were transiently transfected with plasmids containing full-length wild-type VWF or 6 naturally occurring VWF variants located throughout the A domains of VWF. The following mutations, originally identified in type 1 VWD patients, were studied: p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His (A1 domain), p.Tyr1584Cys, p.Arg1583Trp (A2 domain) and p.Val1822Gly (A3 domain). Medium and lysates of transfected cells were collected to measure basal VWF secretion. Transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the regulated VWF secretion. Confocal- and Transmission electron microscopy (TEM) were used to study the formation of WPB. Results: Cells transfected with WT-VWF or VWF p.Arg1583Trp formed numerous elongated pseudo-WPB as evidenced by confocal microscopy. p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His, p.Tyr1584Cys and p.Val1822Gly were able to form these organelles, but in most cases the pseudo-WPB were shorter and more round compared to those formed by WT-VWF. Retention of VWF in the ER was present in about 50% of the cells expressing p.Leu1307Pro and 25–35% of the cells expressing p.Ser1285Pro and p.Val1822Gly as compared to 10% in WT-VWF. The ER retention in p.Arg1374His, p.Tyr1584Cys and p.Arg1583Trp variants was comparable with WT-VWF. Upon co-transfection with WT-VWF the defective elongation of pseudo-WPB was partly corrected. WPB formation was further studied using TEM. In WT-VWF transfected cells, elongated and electron dense pseudo-WPB were observed. p.Arg1583Trp showed cigar-shaped pseudo-WPB with typical VWF striations. Shorter and more round pseudo-WPB were found in cells expressing the VWF variants p.Ser1285Pro, p.Arg1374His, p.Tyr1584Cys. The p.Val1822Gly variant showed some elongated pseudo-WPB, although most of the structures were round. The round organelles are, however, recognizable as pseudo-WPB as they contain tubular structures indicating storage of VWF tubules. The p.Leu1307Pro and p.Val1822Gly showed reduced VWF basal secretion, even in the heterozygous state. Both mutations, together with p.Ser1285Pro, also showed impaired regulated secretion of VWF in both single and co-transfections with wt-VWF. Conclusion: Our data shows that naturally occurring VWD variants within the A domains of VWF can cause defects in both WPB formation and (regulated) secretion of VWF. This is in contrast with previous data which suggests that only the propeptide and D1'-A1 domain of VWF are essential for normal WPB formation. In our study however, we found that two mutations located within the A2 and A3 domain (p.Tyr1584Cys and p.Val1822Gly) also interfere with the formation of elongated WPB as evidenced by round storage organelles containing VWF tubules. Defects in WPB formation and regulated secretion of VWF may be the underlying cause of the poor response to DDAVP infusion seen in a subset of VWD type 1 patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 263 In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation. To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell. In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures: No relevant conflicts of interest to declare.

Abstract 325 The liver is generally recognized as the major site of coagulation factor (F)VIII synthesis. However, there is now increasing evidence that FVIII can also be synthesized in specific endothelial cells where it is stored with its natural carrier protein von Willebrand factor (VWF) in the Weibel-Palade bodies (WPBs). WPBs have a typical cigar-shaped appearance that most likely originates from the macromolecular organization of VWF multimers into tubules. The tubular storage of VWF is thought to be essential for orderly secretion of VWF strings during activation of endothelial cells. Recently we have shown that expression of FVIII with VWF changes the WPB morphology to spherical vesicles. This finding suggests alterations in the biochemical properties of stored VWF. We now studied in detail the effect of FVIII co-expression on the VWF molecule using a combination of innovative techniques, including correlative light-electron microscopy (CLEM), and live-cell fluorescence microscopy under flow conditions. Analysis of human blood outgrowth endothelial cells (BOECs) expressing human B-domain deleted FVIII-GFP by CLEM revealed that FVIII containing WPBs were electron-dense, spherical structures. These structures contained disorganized short VWF tubules, which was confirmed in 3D by electron tomography. Double immunogold labelling with VWF and GFP antibodies showed that the spherical FVIII containing structures were always positive for VWF. These observations imply that FVIII blocks the expansion of VWF tubules, possibly by binding to the N-terminal VWF domains. As the N-terminal domains are also implicated in the formation of multimers, we therefore investigated whether FVIII affects VWF multimer size. Indeed, multimer analysis showed that VWF secreted by FVIII-GFP transduced BOECs was multimerized to a lesser extent when compared to VWF secreted by non-transduced BOECs. The combined absence of high molecular weight (HMW) VWF multimers and long VWF tubules made us question whether these cells could still release ultra-large VWF (UL-VWF) strings. UL-VWF strings play a key role in bleeding arrest, as platelets adhere to the released VWF string which ultimately leads to the formation of a platelet plug. We examined the release of UL-VWF strings under shear stress from BOECs expressing FVIII-GFP employing live-cell confocal imaging. This technique allowed us to follow FVIII release during exocytosis of WPBs in real-time as well. When we stimulated FVIII-transduced BOECs with histamine, these cells were equally able to release VWF strings as non-transduced BOECs. Although spherical WPBs lacked long VWF tubules and did not secrete HMW multimers, released VWF strings were of similar length as strings secreted by non-transduced BOECs. Surprisingly, released VWF strings were completely covered with FVIII which remained attached to the strings throughout the whole experiment. Another remarkable observation was that platelet binding to the FVIII-covered VWF strings was almost completely absent. We hypothesize that FVIII either shields the A1 domain for platelet binding or causes a conformational change in the VWF strings that prevents platelets from binding to the strings. Our results demonstrate that FVIII co-trafficking with VWF has a major impact on properties of VWF as it reduces the degree of multimerization, shortens tubules and prevents platelets from adhering to strings. This leads us to the conclusion that the macromolecular structure of VWF is considerably altered when FVIII is present in WPBs. Disclosures: No relevant conflicts of interest to declare.

This article makes the method of seasonal adjustment operational using suitable structural time series models (STM). This so-called STM method is applied to several relevant Dutch macro- economic quarterly and monthly time series. The results are compared with those of the Census X-11 method using several formal criteria as yardsticks. The STM method proves to compete well with the Census X-11 method in this respect.

1. 1. Oxygen uptake and 14 CO 2 production by goldfish red muscle mitochondria in the presence of various substrates were examined. 2. 2. Isolated mitochondria demonstrate a high efficiency of oxidative phosphorylation and respiratory control ratios ranging from 3.6 to 14.6. 3. 3. In the presence of citrate, isocitrate, fumarate or NADH the respiratory rate is not stimulated by added ADP. 4. 4. Pyruvate is oxidized without addition of a sparker metabolite. 5. 5. No activity of the glycerol phosphate shuttle could be induced. 6. 6. Anoxia and ADP stimulate 14 CO 2 production from [1- 14 C] pyruvate, [2- 14 C] pyruvate and [U- 14 C] glutamate. 7. 7. It is suggested that the regulation of the pyruvate dehydrogenase complex activity is modified during anoxia.

Binding of parathion and paraoxon to bovine serum albumin (BSA) and human serum albumin (HSA) was studied by using equilibrium dialysis. The concentration of unbound organophosphate was determined from its anticholinesterase activity. Binding of parathion to BSA was shown to be reversible. The organophosphates interact with only one type of binding sites in BSA and HSA. The affinity constants at pH 7.2 and 4 degrees C for the interaction of BSA or HSA and parathion were found to be 2.7 X 10(6) and 1.5 X 10(6) M-1, respectively. The affinity constants for the interaction of the serum albumins and paraoxon were considerably lower, 6.0 X 10(3) and 1.6 X 10(4) M-1, respectively. Lowering the pH from 7.2 to 4.8 did not significantly affect the binding parameters. The great difference of affinity of the serum albumins to parathion and paraoxon is discussed with respect to the fate of parathion in the body.

Summary Using a direct macrophage migration inhibition test the hypersensitivity against encephalitogenic protein and phytohaemagglubinin in normal persons, multiple sclerosis patients and patients with other diseases of the central nervous system were examined. It proved that the vast majority of patients were sensitised to brain antigen. The percentage of positive tests and the percentage of migration inhibition was related to the activity of the disease. No differences were found between lymphocytes of multiple sclerosis patients and of patients with the other neurological diseases patients. Foetal calf serum was proven to depress the hypersensitivity to phytohaemagglutinin as did multiple sclerosis serum on normal lymphocytes. The results did not support the hypothesis that multiple sclerosis is caused by a cell-mediated auto-immune proces.

The primary cellular immune responses in multiple sclerosis (MS) patients as compared with healthy controls were investigated with alpha-HPH and DNCB sensitization tests. The results did not reveal significant differences, indicating that no defect or hyperactive state of the cellular immune system is present in MS patients. Some minor and not significant differences between groups of patients or controls could be related to age differences and the degree of invalidism.