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This multi-arm, parallel group, single-blinded randomised controlled trial aimed to assess three commercially available mouthwashes effectiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This manuscript has been written in accordance with the CONSORT statement. Methods. Eligible participants were SARS-CoV-2 positive with a positive test in the last 72 h. All participants had mild to moderate symptoms and could provide five saliva samples over a 60 min period. Participants delivered a baseline saliva sample and then used a mouthwash as per manufacturer's instructions. They provided further saliva samples at minute 1, 10, 30 and 60. Participants were randomised to one of four groups; OraWize+, Total Care Listerine, Cool Mint Listerine and water (control). The lab-based research team were blind to the intervention. The research question was: can SARS-CoV-2 be rendered inactive in saliva by using a mouthwash and how long does this effect last? The primary outcome was the amount of viable infectious SARS-CoV-2 virus in the sample, compared to the baseline sample. The secondary outcome measure was the amount of genetic material from the SARS-CoV-2 virus in the sample, measured via PCR testing. Results. In total 100 participants were recruited (25 per group). Eight participants did not receive the allocated intervention and did not have saliva samples collected. There were no adverse events. In total 42 of the 92 participants had viable virus which could be cultured at baseline. Statistical analysis of the primary outcome was not advised due to the reduced level of viable virus at baseline and the positive skewness present in the distribution of log10(titre) data. Observational data of the primary outcome measure is presented. Analysis of the secondary outcome PCR measure showed that there was strong evidence for a decrease in SARS-CoV-2 RNA levels compared to water for all mouthwashes after 1 min, OraWize+ -0.49 (-0.92, -0.05),  p -value 0.029, Cool Mint Listerine -0.81 (-1.25, -0.38),  p -value<0.001, Total Care Listerine -1.05 (-1.48, -0.62),  p -value<0.001. For the remaining timepoints there was generally no evidence of virus level reduction compared to water although there is weak evidence for a decrease at ten minutes using Total Care Listerine -0.44 (-0.88, 0.01), p -value 0.053. Conclusion. The three mouthwashes included in this trial observationally demonstrated a reduction in virus titre level 1 min after use, with virus levels normalising up to 60 min compared to the control. Although an interesting observation, this result could not be statistically analysed. Using the secondary outcome PCR measure all three included mouthwashes reduced virus levels compared to water at 1 min and these results were statistically significant. Clinically this result does not support the use of the included mouthwashes to reduce SARS-CoV-2 levels in saliva., Competing Interests: There were no conflicts of interest., (Copyright © 2024 The Authors.)

The assembly of von Willebrand factor (VWF) into ordered helical tubules within endothelial Weibel-Palade bodies (WPBs) is required for the efficient deployment of the protein at sites of vascular injury. VWF trafficking and storage are sensitive to cellular and environmental stresses that are associated with heart disease and heart failure. Altered storage of VWF manifests as a change in WPB morphology from a rod shape to a rounded shape and is associated with impaired VWF deployment during secretion. In this study, we examined the morphology, ultrastructure, molecular composition and kinetics of exocytosis of WPBs in cardiac microvascular endothelial cells isolated from explanted hearts of patients with a common form of heart failure, dilated cardiomyopathy (DCM; HCMEC D ), or from nominally healthy donors (controls; HCMEC C ). Using fluorescence microscopy, WPBs in HCMEC C (n = 3 donors) showed the typical rod-shaped morphology containing VWF, P-selectin and tPA. In contrast, WPBs in primary cultures of HCMEC D (n = 6 donors) were predominantly rounded in shape and lacked tissue plasminogen activator (t-PA). Ultrastructural analysis of HCMEC D revealed a disordered arrangement of VWF tubules in nascent WPBs emerging from the trans-Golgi network. HCMEC D WPBs still recruited Rab27A, Rab3B, Myosin-Rab Interacting Protein (MyRIP) and Synaptotagmin-like protein 4a (Slp4-a) and underwent regulated exocytosis with kinetics similar to that seen in HCMECc. However, secreted extracellular VWF strings from HCMEC D were significantly shorter than for endothelial cells with rod-shaped WPBs, although VWF platelet binding was similar. Our observations suggest that VWF trafficking, storage and haemostatic potential are perturbed in HCMEC from DCM hearts.

In response to vascular damage, P-selectin molecules are secreted onto the surface of cells that line our blood vessels. They then serve as mechanical anchors to capture leucocytes from the blood stream. Here, we track individual P-selectin molecules released at the surface of live endothelial cells following stimulated secretion. We find P-selectin initially shows fast, unrestricted diffusion but within a few minutes, movement becomes increasingly restricted and ~50% of the molecules become completely immobile; a process similar to a sol-gel transition. We find removal of the extracellular C-type lectin domain (ΔCTLD) and/or intracellular cytoplasmic tail domain (ΔCT) has additive effects on diffusive motion while disruption of the adapter complex, AP2, or removal of cell-surface heparan sulphate restores mobility of full-length P-selectin close to that of ΔCT and ΔCTLD respectively. We have found P-selectin spreads rapidly from sites of exocytosis and evenly decorates the cell surface, but then becomes less mobile and better-suited to its mechanical anchoring function., (© 2022. The Author(s).)

A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures., (© 2021. The Author(s).)

The long-term effects of climate change on biodiversity and biogeographic patterns are uncertain. There are known relationships between geographic area and both the number of species and the number of ecological functional groups-termed the species-area relationship and the functional diversity-area relationship, respectively. We show that there is a positive relationship between the number of species in an area, the number of ecological functional groups, and oceanic temperature in the shallow-marine fossil record of New Zealand over a time span of ~40 million years. One implication of this relationship is that functional redundancy increases with temperature. This reveals a long-lived and persistent association between the spatial structuring of biodiversity, the temperature-dependence of functional redundancy, and shallow-marine biodiversity in mid-latitudes., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens., (© Crown copyright 2020.)

In this study, we present 77 transmission electron microscopy (TEM) images of human brainstem tissue from 11 cases of late onset Parkinson's disease (PD). The tissues were fixed, embedded, sectioned, and stained for TEM application. In addition, we present 11 images from autopsy specimens of 1 case of human poliomyelitis infection as positive controls and 12 images from 8 cases of autopsy specimens of other conditions as negative controls. In the TEM images of the PD cases there were cytoplasmic inclusion bodies consisting of virus-like particles (VLP) 30 nm in diameter that were associated with endoplasmic reticulum membranes.  In the nuclei of the PD neurons there were VLP ranging from 40 nm to 50 nm in diameter. In the poliomyelitis cases, similar particles as were observed in PD which were interpreted to be poliomyelitis virus particles. In the negative controls one case was identified which showed similar VLP (Figure 1, controls).  A Lewy body was found in this "control" case (Figure 10) suggesting that this was an undiagnosed case of PD. Cytoplasmic ribosomes measuring approximately 17 nm were observed in the control neurons., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Dourmashkin RR et al.)

Elevations of intracellular free Ca 2+ concentration ([Ca 2+ ] i ) are a potent trigger for Weibel-Palade body (WPB) exocytosis and secretion of von Willebrand factor (VWF) from endothelial cells; however, the identity of WPB-associated Ca 2+ -sensors involved in transducing acute increases in [Ca 2+ ] i into granule exocytosis remains unknown. Here, we show that synaptotagmin 5 (SYT5) is expressed in human umbilical vein endothelial cells (HUVECs) and is recruited to WPBs to regulate Ca 2+ -driven WPB exocytosis. Western blot analysis of HUVECs identified SYT5 protein, and exogenously expressed SYT5-mEGFP localised almost exclusively to WPBs. shRNA-mediated knockdown of endogenous SYT5 (shSYT5) reduced the rate and extent of histamine-evoked WPB exocytosis and reduced secretion of the WPB cargo VWF-propeptide (VWFpp). The shSYT5-mediated reduction in histamine-evoked WPB exocytosis was prevented by expression of shRNA-resistant SYT5-mCherry. Overexpression of SYT5-EGFP increased the rate and extent of histamine-evoked WPB exocytosis, and increased secretion of VWFpp. Expression of a Ca 2+ -binding defective SYT5 mutant (SYT5-Asp197Ser-EGFP) mimicked depletion of endogenous SYT5. We identify SYT5 as a WPB-associated Ca 2+ sensor regulating Ca 2+ -dependent secretion of stored mediators from vascular endothelial cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: In a previous study on encephalitis lethargica, we identified a virus related to enterovirus in autopsy brain material. Transmission electron microscopy (TEM), immunohistochemistry (IHC) and molecular analysis were employed.  Our present objective was to investigate, using a similar approach, as to whether virus-like particles (VLP) and enterovirus antigen are present in Parkinson's disease (PD) brainstem neurons. Methods: Fixed tissue from autopsy specimens of late onset PD and control brainstem tissue were received for study. The brain tissue was processed for TEM and IHC according to previous published methods. Results:  We observed VLP in the brainstem neurons of all the cases of PD that were examined.  In the neurons' cytoplasm there were many virus factories consisting of VLP and endoplasmic reticulum membranes. In some neurons, the virus factories contained incomplete VLP. Complete VLP in some neurons' virus factories had an average diameter of 31 nm, larger than control brain ribosomes. In the nuclei, there were VLP with an average diameter of 40 nm. In cases of human poliomyelitis, there were cytoplasmic virus factories and intranuclear virus particles similar to those observed in PD. On preparing PD brain sections for IHC there was positive staining using anti-poliovirus antibody and anti-coxsackie antibody. This result was statistically significant. Conclusions: We present evidence for an enterovirus infection in PD.  For future studies, virus isolation and molecular analysis are suggested., Competing Interests: No competing interests were disclosed.

Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa., (© 2016. Published by The Company of Biologists Ltd.)

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis.

Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.

In New Zealand, an arbovirus surveillance program has been operating for more than 20 years, which includes testing of cattle with the Akabane virus neutralization test. With the aim to replace this laborious test by an easier-to-perform enzyme-linked immunosorbent assay (ELISA), 2 commercial ELISA kits, ELISA-1 from France (originally from Australia) and ELISA-2 from Japan, were compared, using 334 serum samples from noninfected New Zealand cattle, and 548 serum samples from naturally infected cattle herds in Australia. Diagnostic specificities for the test methods were high, ranging from 99.4% to 100%. The diagnostic sensitivities varied considerably between the test methods and differed from the values reported by the manufacturers (94% for each ELISA). The diagnostic sensitivities relative to the virus neutralization test (n = 378) were 96.0% for ELISA-1 or 98.9% when suspect samples were included, and 78.0% for ELISA-2. Differences in the commercial ELISA kits may be explained by the presence of other Simbu serogroup viruses in Australian cattle herds, causing cross-reactions in ELISA-1. Both commercial ELISA kits would be fit for purpose and could replace the virus neutralization test for Akabane virus surveillance in New Zealand. ELISA-1 may be able to detect other Simbu serogroup viruses, should they be present. The current study shows that despite comparable ELISA test characteristics given by the manufacturers, evaluation on the target population revealed marked differences in the ELISA kits test methods' characteristics.

Background: The mechanisms of antibody-mediated damage to allografts are not well understood. We have examined the effect of antibodies to human leukocyte antigens on secretion of von Willebrand factor (vWF) from endothelial cells (ECs)., Methods: The effect of monoclonal antibodies (W6/32, L2, and L243), in the presence and absence of sublytic concentrations of complement, on the release of vWF from Weibel-Palade bodies (WPBs) in human umbilical vein ECs (HUVECs), human aortic ECs (HAECs), and human heart microvascular ECs (HHMECs) was investigated using biochemical and live-cell imaging. Fura-2-loaded ECs expressing the WPB marker proregion-enhanced green fluorescence protein were imaged simultaneously for intracellular Ca(2+) changes ([Ca(2+)](i)) and WPB exocytosis., Results: Stimulation of ECs with 1- or 10-µg/mL W6/32, L2, or L243 did not evoke significant vWF release above control IgG. In live-cell imaging studies, exposure of proregion-enhanced green fluorescence protein-expressing HAECs to physiologic saline, 10-µg/mL U9F4, or W6/32 alone for 5 to 10 min induced irregular (Ca(2+))(i)\ spiking but no WPB exocytosis. Histamine-evoked WPB exocytosis was not changed by preexposure of HAECs to physiologic saline, U9F4, or W6/32. Stimulation of HUVECs with sublytic complement concentrations evoked WPB exocytosis; however, the addition of W6/32 did not change the amount of vWF released., Conclusion: Antibodies to human leukocyte antigen class I or II do not elicit significant WPB exocytosis or vWF secretion from ECs in the absence of exogenous complement.

Weibel-Palade body (WPB) exocytosis underlies hormone-evoked VWF secretion from endothelial cells (ECs). We identify new endogenous components of the WPB: Rab3B, Rab3D, and the Rab27A/Rab3 effector Slp4-a (granuphilin), and determine their role in WPB exocytosis. We show that Rab3B, Rab3D, and Rab27A contribute to Slp4-a localization to WPBs. siRNA knockdown of Slp4-a, MyRIP, Rab3B, Rab3D, Rab27A, or Rab3B/Rab27A, or overexpression of EGFP-Slp4-a or EGFP-MyRIP showed that Slp4-a is a positive and MyRIP a negative regulator of WPB exocytosis and that Rab27A alone mediates these effects. We found that ECs maintain a constant amount of cellular Rab27A irrespective of the WPB pool size and that Rab27A (and Rab3s) cycle between WPBs and a cytosolic pool. The dynamic redistribution of Rab proteins markedly decreased the Rab27A concentration on individual WPBs with increasing WPB number per cell. Despite this, the probability of WPB release was independent of WPB pool size showing that WPB exocytosis is not determined simply by the absolute amount of Rab27A and its effectors on WPBs. Instead, we propose that the probability of release is determined by the fractional occupancy of WPB-Rab27A by Slp4-a and MyRIP, with the balance favoring exocytosis.

New Zealand is free from equine influenza and has never experienced an incursion in its horse population. As part of New Zealand's preparedness to an incursion of an exotic animal disease, it was considered necessary to select the most accurate test for equine influenza (EI) from the array of those available. Four readily available blocking/competitive enzyme-linked immunosorbent assays (ELISA), originally developed and marketed for the detection of antibodies against the avian influenza virus, were evaluated using serum samples from New Zealand non-infected, non-vaccinated horses (n=365), and Australian field infected (n=99) and experimentally infected horses (n=3). Diagnostic specificities (DSP) and diagnostic sensitivities (DSE) were determined as follows: ELISA-1=98.1%/99.0%; ELISA-2=90.1%/99.0%; ELISA-3=98.1%/96.0%; ELISA-4=95.3%/99.0%. For ELISA-1, DSP and DSE results were comparable to previously published data on a larger sample number from Australian horses (Sergeant et al., 2009). Receiver operating characteristics (ROC) and frequency histogram analysis were also performed. The area under the curve (AUC) ranged from 0.996 to 0.979, with ELISA-1 possessing the highest AUC, followed by ELISA-2, ELISA-4 and ELISA-3. Separation of the negative and the positive serum panel was best for ELISA-4, followed by ELISA-2, ELISA-1 and ELISA-3. In three experimentally infected horses, sero-positivity was detected between 7 and 9 days post-infection, with ELISA-4 being most sensitive, followed by ELISA-1, ELISA-2 and ELISA-3. Overall, the four ELISAs performed well in this evaluation but some differences were observed., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca(2+)-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca(2+)-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs., Principal Findings: Ca(2+)-driven WPB exocytosis occurred at all temperatures studied (7-37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s(-1) (37°C) to 0.80±0.14 s(-1) (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca(2+)-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75-80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60-120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself., Conclusions: We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased.

In a consignment of sheep brains from New Zealand, to be used in Europe as negative control material in scrapie rapid screening test evaluations, brain samples from 1 sheep (no. 1512) gave the following initially confusing results in various screening tests: the brainstem repeatedly produced negative results in 2 very similar screening kits (enzyme-linked immunosorbent assay [ELISA]-1, ELISA-2), a macerate made from brainstem and cerebellum returned a clearly positive result in ELISA-2, and the macerate and a brainstem sample gave negative results in a third screening test (ELISA-3). In subsequent testing, cerebellum tissue alone tested strongly positive in ELISA-1 and produced a banding pattern very similar to atypical scrapie/Nor98 in a confirmatory Western blot (WB). The macerate showed weak staining in the confirmatory WB but presented a staining pattern identical to atypical scrapie/Nor98 in the scrapie-associated fibril WB. The latter test confirmed conclusively the first case of atypical scrapie/Nor98 in a sheep from New Zealand. Other parts of the brain either tested negative or very weak positive in ELISA-2 and in WBs, or tested with negative results by histopathology and immunohistochemistry. It appears that sheep no. 1512 is a case of atypical scrapie/Nor98 in which the abnormal prion protein was detected mainly in the cerebellum. This case emphasizes the need to retain brainstem, and cerebral and cerebellar tissues, as frozen and fixed materials, for conclusive confirmatory testing. Furthermore, consideration should be given to which screening method to use.

Endothelial cells are reported to contain several distinct populations of regulated secretory organelles, including Weibel-Palade bodies (WPBs), the tissue plasminogen activator (tPA) organelle, and the type-2 chemokine-containing organelle. We show that the tPA and type-2 organelles in human endothelial cells represent a single compartment primarily responsible for unstimulated secretion of tPA or, in cells exposed to interleukin-1β (IL-1β), the cytokines IL-8, IL-6, monocyte chemoattractant protein-1 (MCP-1), and growth-regulated oncogene-α (GRO-α). This compartment was distinct from WPBs in that it lacked detectable von Willebrand factor, P-selectin, Rab27a, or CD63 immunoreactivity, displayed no time-dependent decrease in intragranule pH, underwent detectable unstimulated exocytosis, and was very poorly responsive to Ca(2+)-elevating secretagogues. WPBs could also contain tPA, and in IL-1β-treated cells, IL-8, IL-6, MCP-1, and GRO-α, and were the primary source for histamine or ionomycin-stimulated secretion of these molecules. However, analysis of the storage efficiency of cytokines and tPA revealed that all were very poorly stored compared with von Willebrand factor. The nonmammalian, nonsecretory protein EGFP, when expressed in the secretory pathway, also entered WPBs and had a storage efficiency similar to tPA and the cytokines tested. Based on these data, we proposed a revised model for storage and secretion of cytokines and tPA.

Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.

Aim: To make valid recommendations on the use of serological test methods for the detection of serum antibodies in ruminants against Coxiella burnetii (Q-fever), by comparing the performance of the complement fixation test (CFT) and two ELISA, and by identifying reasons for discrepancies between the test methods., Methods: A total of 73 serum samples from infected cattle, 69 from infected goats, and 100 samples from non-infected cattle and 57 samples from non-infected sheep, as well as 95 samples from infected cattle herds (mix of seropositive and seronegative samples), were tested using the CFT, the IDEXX ELISA (I-ELISA) and the Pourquier ELISA (P-ELISA). A mixed panel of 12 serum samples from sheep from inter-laboratory proficiency testing (proficiency panel) was also tested using the CFT and both ELISA, and further investigated using IgG- and IgM-specific ELISA., Results: Generally, the two commercial ELISA were more sensitive than the CFT for the detection of infected ruminants. Good agreement between ELISA for positive and negative results was found for samples from the infected herd, while results for the positive panels varied between the two ELISA. For the total of the positive serum panels, the I-ELISA detected 95% of samples as positive or suspicious, while the P-ELISA detected only 81%. In the P-ELISA, more samples were considered suspicious (18%) than in the I-ELISA (14%). All sera from non-infected sheep and cattle tested negative in the serological test methods employed, except for one positive sample from a sheep in the P-ELISA. Further investigation revealed that a CFT-positive but ELISA-negative result was due to high IgM and low IgG reactivity., Conclusions: The two commercial ELISA were more sensitive than the CFT in all panels from infected ruminants. However, they could only detect IgG. The I-ELISA should be the serological test method of choice for cattle, sheep and goats for import testing of animals into New Zealand because it was more sensitive than the P-ELISA and was equally specific to the PELISA and the CFT. For other animal species, such as deer and camelids, the CFT should still be used since none of the ELISA has been evaluated for these species. This study has shown that the two commercial ELISA will detect the majority of infected ruminants but may miss animals that have not developed an IgG response.

GH has physiological functions in many tissues, but the cellular targets for direct effects of GH remain ill defined in complex tissues such as the growth plate in which the contribution of direct vs. indirect actions of GH remains controversial. The Janus kinase (Jak)-signal transducer and activator of transcription (STAT)-5 pathway is activated by GH, so we developed a method to visualize nuclear Stat5b and phosphorylated Stat5 in single cells in response to a pulse of GH. Hep2 cells did not show a Stat5 phosphorylation (pY-Stat5) response to GH except in cells transfected to express GH receptors. ATDC5 cells express GH receptors and showed GH-induced pY-Stat5 responses, which varied with their state of chondrocyte differentiation. In vivo, Stat5b(+ve) nuclei were seen in the resting and prehypertrophic chondrocytes of the growth plate. After a single ip pulse of human GH or mouse GH, but not prolactin, pY-Stat5 responses were visible in cells in the resting zone and groove of Ranvier, 10-45 min later. Prehypertrophic chondrocytes showed no pY-Stat5 response to GH. GH target cells were also identified in other tissues, and a marked variability in spatiotemporal pY-Stat5 responses was evident. Endogenous hepatic pY-Stat5 was detected in mice with intact GH secretion but only during a GH pulse. Fasting and chronic exposure to GH attenuated the pY-Stat5 response to an acute GH injection. In conclusion, pY-Stat5 responses to GH vary in time and space, are sensitive to nutritional status, and may be inhibited by prior GH exposure. In the growth plate, our data provide direct in vivo support for an early role of GH to regulate the fate of immature chondrocytes.

Proteins secreted from Weibel-Palade bodies (WPBs) play important roles in regulating inflammatory and hemostatic responses. Inflammation is associated with the extracellular acidification of tissues and blood, conditions that can alter the behavior of secreted proteins. The effect of extracellular pH (pH(o)) on the release of von Willebrand factor (VWF), the VWF-propolypeptide (Proregion), interleukin-8, eotaxin-3, P-selectin, and CD63 from WPBs was investigated using biochemical approaches and by direct optical analysis of individual WPB fusion events in human endothelial cells expressing green or red fluorescent fusions of these different cargo proteins. Between pH(o) 7.4 and 7.0, ionomycin-evoked WPB exocytosis was characterized by the adhesion of VWF to the cell surface and the formation of long filamentous strands. The rapid dispersal of Proregion, interleukin-8, and eotaxin-3 into solution, and of P-selectin and CD63 into the plasma membrane, was unaltered over this pH(o) range. At pH(o) 6.8 or lower, Proregion remained associated with VWF, in many cases WPB failed to collapse fully and VWF failed to form filamentous strands. At pH(o) 6.5 dispersal of interleukin-8, eotaxin-3, and the membrane protein CD63 remained unaltered compared with that at pH(o) 7.4; however, P-selectin dispersal into the plasma membrane was significantly slowed. Thus, extracellular acidification to levels of pH(o) 6.8 or lower significantly alters the behavior of secreted VWF, Proregion, and P-selectin while rapid release of the small pro-inflammatory mediators IL-8 and eotaxin-3 is essentially unaltered. Together, these data suggest that WPB exocytosis during extracellular acidosis may favor the control of inflammatory processes.

Endothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.

The small GTP-binding protein Ral has been implicated in regulated exocytosis via its interaction with the mammalian exocyst complex. We have previously demonstrated that Ral is involved in exocytosis of Weibel-Palade bodies (WPBs). Little is known about intracellular signaling pathways that promote activation of Ral in response to ligand binding of G protein-coupled receptors. Here we show that RNAi-mediated knockdown of RalGDS, an exchange factor for Ral, results in inhibition of thrombin- and epinephrine-induced exocytosis of WPBs, while overexpression of RalGDS promotes exocytosis of WPBs. A RalGDS variant lacking its exchange domain behaves in a dominant negative manner by blocking release of WPBs. We also provide evidence that RalGDS binds calmodulin (CaM) via an amino-terminal CaM-binding domain. RalGDS association to CaM is required for Ral activation because a cell-permeable peptide comprising this RalGDS CaM-binding domain inhibits Ral activation and WPB exocytosis. Together our findings suggest that RalGDS plays a vital role in the regulation of Ral-dependent WPB exocytosis after stimulation with Ca(2+)- or cAMP-raising agonists.

Exocytosis of specialized endothelial cell secretory organelles, Weibel-Palade bodies (WPBs), is thought to play an important role in regulating hemostasis and intravascular inflammation. The major WPB core proteins are Von Willebrand factor (VWF) and its propolypeptide (Proregion), constituting more than 95% of the content. Although the composition of the WPBs can be fine-tuned to include cytokines and chemokines (eg, interleukin-8 [IL-8] and eotaxin-3), it is generally assumed that WPB exocytosis is inextricably associated with secretion of VWF. Here we show that WPBs can undergo a form of exocytosis during which VWF and Proregion are retained while smaller molecules, such as IL-8, are released. Imaging individual WPBs containing fluorescent cargo molecules revealed that during weak stimulation approximately 25% of fusion events result in a failure to release VWF or Proregion. The WPB membrane protein P-selectin was also retained; however, the membrane tetraspannin CD63 was released. Accumulation or exclusion of extracellular fluorescent dextran molecules ranging from 3 kDa to 2 mDa show that these events arise due to the formation of a fusion pore approximately 12 nm in diameter. The pore behaves as a molecular filter, allowing selective release of WPB core and membrane proteins. WPB exocytosis is not inextricably associated with secretion of VWF.

The rate, concentration dependence and extent of histamine-evoked Weibel-Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45+/-0.26 (s.d., n=144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0+/-4.42 s (n=9 cells) at 0.3 microM histamine and 1.57+/-0.21 s (n=15 cells) at 100 microM histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20+/-0.16 WPB s(-1) (n=9) at 0.3 microM and 3.66+/-0.45 WPB s(-1) at 100 microM histamine (n=15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 microM), for which there was a delay between calcium elevation and secretion of 1.67+/-0.24 s (n=6), and a peak fusion rate of approximately 10 WPB s(-1).

The biogenesis of endothelial-specific Weibel-Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3-dependent (AP-3-dependent), and AP-3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P-selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP-3, which operates in post-Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P-selectin is recruited to forming WPB in the trans-Golgi by AP-3-independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already-budded WPB by an AP-3-dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).

The time course for cell surface loss of von Willebrand factor (VWF) and the propolypeptide of VWF (proregion) following exocytosis of individual Weibel-Palade bodies (WPBs) from single human endothelial cells was analyzed. Chimeras of enhanced green fluorescent protein (EGFP) and full-length pre-pro-VWF (VWF-EGFP) or the VWF propolypeptide (proregion-EGFP) were made and expressed in human umbilical vein endothelial cells. Expression of VWF-EGFP or proregion-EGFP resulted in fluorescent rod-shaped organelles that recruited the WPB membrane markers P-selectin and CD63. The WPB secretagogue histamine evoked exocytosis of these fluorescent WPBs and extracellular release of VWF-EGFP or proregion-EGFP. Secreted VWF-EGFP formed distinctive extracellular patches of fluorescence that were labeled with an extracellular antibody to VWF. The half-time for dispersal of VWF-EGFP from extracellular patches was 323.5 +/- 146.2 s (+/-S.D., n = 20 WPBs). In contrast, secreted proregion-EGFP did not form extracellular patches but dispersed rapidly from its site of release. The half-time for dispersal of proregion-EGFP following WPB exocytosis was 2.98 +/- 1.88 s (+/-S.D., n = 32 WPBs). The slow rate of loss of VWF-EGFP is consistent with the adhesive nature of this protein for the endothelial membrane. The much faster rate of loss of proregion-EGFP indicates that this protein does not interact strongly with extracellular VWF or the endothelial membrane and consequently may not play an adhesive role at the endothelial cell surface.

The identification of organelles is crucial for efficient cellular function, yet the basic underlying mechanisms by which this might occur have not been established. One group of proteins likely to be central to organelle identity is the Rab family of small GTPases. We have thus investigated Rab recruitment to membranes using endothelial cells as a model system. We report that Weibel-Palade bodies, the Von Willebrand Factor storage compartment of human umbilical vein endothelial cells, contain Rab27a. We have also found that Weibel-Palade body-like structures induced in HEK-293 cells by the expression of von Willebrand factor can recruit endogenous Rab27a. In the absence of von Willebrand Factor, Rab27a is not lysosome associated, indicating that it can distinguish between the Weibel-Palade-body-like organelle and a classical lysosome. Finally, a time course of Weibel-Palade-body formation was established using a green-fluorescent version of von Willebrand factor. Newly formed Weibel-Palade bodies lack Rab27a, which is acquired some hours after initial appearance of the cigar-shaped organelle. We conclude that a lumenal cargo protein drives the recruitment of Rab27a to the organelle membrane by a novel mechanism that is indirect, maturation-dependent and cell-type independent.

Weibel-Palade bodies (WPBs) are the lysosome-related secretory organelles of endothelial cells. Their content protein von Willebrand factor, plays a key role in haemostasis, whilst P-selectin in the membranes is critical in the initiation of inflammation. Biogenesis of these rod-shaped structures is driven by von Willebrand factor, since its heterologous expression leads to formation of organelles morphologically indistinguishable from bona fide WPBs. The two main membrane proteins of WPBs, CD63 and P-selectin, have complex itineraries controlled largely by cytoplasmic targeting signals. We are only just beginning to understand the way in which these three proteins come together to form mature WPBs.

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule, are uncharacterized. We have examined the ability of these pseudogranules, which we show are secretagogue responsive, to recruit membrane proteins. Coexpression experiments show that the Weibel-Palade body proteins P-selectin and CD63, as well as the secretory organelle membrane proteins vesicle-associated membrane protein-2 and synaptotagmin I are diverted away from the endogenous adrenocorticotropic hormone-containing secretory granules to the vWF-containing pseudogranules. However, transferrin receptor, lysosomal-associated membrane protein 1, and sialyl transferase are not recruited. The recruitment of P-selectin is dependent on a tyrosine-based motif within its cytoplasmic domain. Our data show that vWF pseudogranules specifically recruit a subset of membrane proteins, and that in a process explicitly driven by the pseudogranule content (i.e., vWF), the active recruitment of at least one component of the pseudogranule membrane (i.e., P-selectin) is dependent on residues of P-selectin that are cytosolic and therefore unable to directly interact with vWF.

Between 34 and 15 million years (Myr) ago, when planetary temperatures were 3-4 degrees C warmer than at present and atmospheric CO2 concentrations were twice as high as today, the Antarctic ice sheets may have been unstable. Oxygen isotope records from deep-sea sediment cores suggest that during this time fluctuations in global temperatures and high-latitude continental ice volumes were influenced by orbital cycles. But it has hitherto not been possible to calibrate the inferred changes in ice volume with direct evidence for oscillations of the Antarctic ice sheets. Here we present sediment data from shallow marine cores in the western Ross Sea that exhibit well dated cyclic variations, and which link the extent of the East Antarctic ice sheet directly to orbital cycles during the Oligocene/Miocene transition (24.1-23.7 Myr ago). Three rapidly deposited glacimarine sequences are constrained to a period of less than 450 kyr by our age model, suggesting that orbital influences at the frequencies of obliquity (40 kyr) and eccentricity (125 kyr) controlled the oscillations of the ice margin at that time. An erosional hiatus covering 250 kyr provides direct evidence for a major episode of global cooling and ice-sheet expansion about 23.7 Myr ago, which had previously been inferred from oxygen isotope data (Mi1 event).

Here, to study lipid-protein interactions that contribute to the biogenesis of regulated secretory vesicles, we have developed new approaches by which to label proteins in vivo, using photoactivatable cholesterol and glycerophospholipids. We identify synaptophysin as a major specifically cholesterol-binding protein in PC12 cells and brain synaptic vesicles. Limited cholesterol depletion, which has little effect on total endocytic activity, blocks the biogenesis of synaptic-like microvesicles (SLMVs) from the plasma membrane. We propose that specific interactions between cholesterol and SLMV membrane proteins, such as synaptophysin, contribute to both the segregation of SLMV membrane constituents from plasma-membrane constituents, and the induction of synaptic-vesicle curvature.

Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells.

COPI-coated vesicles that bud off the Golgi complex contain two major transmembrane proteins, p23 and p24. We have localized the protein at the Golgi complex and at COPI-coated vesicles. Transport from the intermediate compartment (IC) to the Golgi can be blocked at 15 degrees C, and under these conditions p24 accumulates in peripheral punctated structures identified as IC. Release from the temperature block leads to a redistribution of p24 to the Golgi, showing that p24, similar to p23, cycles between the IC and Golgi complex. Immunoprecipitations of p24 from cell lysates and from detergent-solubilized Golgi membranes and COPI-coated vesicles show that p24 and p23 interact with each other to form a complex. Transient transfection of p23 in HeLa cells shows that p23 and p24 colocalize in structures induced by the overexpression of p23. Taken together p24 interacts with p23 and constitutively cycles between the organelles of the early secretory pathway.

Synaptic vesicles, which have been a paradigm for the fusion of a vesicle with its target membrane, also serve as a model for understanding the formation of a vesicle from its donor membrane. Synaptic vesicles, which are formed and recycled at the periphery of the neuron, contain a highly restricted set of neuronal proteins. Insight into the trafficking of synaptic vesicle proteins has come from studying not only neurons but also neuroendocrine cells, which form synaptic-like microvesicles (SLMVs). Formation and recycling of synaptic vesicles/SLMVs takes place from the early endosome and the plasma membrane. The cytoplasmic machinery of synaptic vesicle/SLMV formation and recycling has been studied by a variety of experimental approaches, in particular using cell-free systems. This has revealed distinct machineries for membrane budding and fission. Budding is mediated by clathrin and clathrin adaptors, whereas fission is mediated by dynamin and its interacting protein SH3p4, a lysophosphatidic acid acyl transferase.

While investigating the localization of synaptophysin in PC12 cells using immunofluorescence microscopy, we noticed a striking difference in its apparent subcellular distribution depending on whether digitonin or Triton X-100 was used as permeabilization agent of paraformaldehyde (PFA)-fixed cells. We found that this difference was due to epitope inaccessibility in the digitonin-treated cells combined with an almost quantitative extraction of the antigen on Triton X-100 permeabilization. Both phenomena were differential with respect to the various synaptophysin-containing compartments. The extraction of antigen from PFA-fixed cells was also seen with other membrane proteins but not with cytosolic proteins and proteins in the lumen of the secretory pathway. Significantly, some of the membrane proteins were extracted from the PFA-fixed cells in higher-molecular-weight forms which we believe represent their in vivo oligomeric states. The implications of our observations are discussed with respect to the method of immunofluorescence microscopy and also to the possible use of paraformaldehyde as an in vivo crosslinker for the study of membrane protein quaternary structure., (Copyright 1998 Academic Press.)

We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

A number of thymic preparations are known to stimulate corticotropin (ACTH) release from pituitary cells but it remains unclear whether this effect is mediated by the corticotropin-releasing hormone (CRH) receptor-associated pathway. We report here that thymosin fraction five (TF5), peptide MB-35 and possibly calf thymus histones can stimulate the release of ACTH from a CRH-insensitive variant of the mouse corticotropic cell line AtT20. The effective concentration range at which TF5 and MB-35 displayed their ACTH-releasing activity in a dose-dependent manner was 100 to 2,000 micrograms/ml and 10 to 100 ng/ml, respectively, whereas neither preparation induced a significant depletion of intracellular ACTH stores. Our data suggest that thymosin peptides can stimulate ACTH release from corticotrophs by a CRH receptor-independent mechanism.

Thymosin fraction five (TF5), a well-characterized immunoregulatory thymic preparation, has been reported to stimulate corticotropin (ACTH) release from rat pituitary cells. Since a previous study in our laboratory had shown that TF5 was able to stimulate ACTH release from corticotropin-releasing hormone (CRH)-insensitive corticotropic tumor cells, it was of interest to assess the role of calcium in the mechanism of action of TF5 on corticotropic cells. A CRH-insensitive variant, denoted AtT-20(CI), of the wild-type corticotropic tumor cell line AtT-20 was used. Synthetic h/rCRH within a dose range of 0.1-100 nM was completely ineffective to stimulate basal ACTH release from AtT-20(CI) cells, although the same batch of neuropeptide displayed the expected ACTH-releasing activity on dispersed rat pituitary cells (for instance, 0.1 nM CRH induced a 3.7-fold increase in ACTH release in this cell system). Median eminence extracts (1/10) induced only a 12% increase in ACTH release from AtT-20(CI) cells as compared to the 395% stimulation induced in normal pituitary cells. As expected, TF5 induced a dose-dependent increase in ACTH release from AtT-20(CI) cells. However, this ACTH-releasing activity of TF5 was completely abolished when cells were incubated in Ca-free medium or Ca-free medium containing 0.5 mM EGTA. On the other hand, the presence of the Ca ionophore A23187 (5 microM) in medium containing normal Ca levels (2.5 mM) did not affect the ACTH-releasing activity of TF5 on AtT-20(CI) cells.(ABSTRACT TRUNCATED AT 250 WORDS)