Granule mobility, fusion frequency and insulin secretion are differentially affected by insulinotropic stimuli.

The pre-exocytotic behavior of insulin granules was studied against the background of the entirety of submembrane granules in MIN6 cells, and the characteristics were compared with the macroscopic secretion pattern and the cytosolic Ca(2+) concentration of MIN6 pseudo-islets at 22°C, 32°C and 37°C. The mobility of granules labeled by insulin-EGFP and the fusion events were assessed by TIRF microscopy utilizing an observer-independent algorithm. In the z-dimension, 40 mm K(+) or 30 mm glucose increased the granule turnover. The effect of high K(+) was quickly reversible. The increase by glucose was more sustained and modified the efficacy of a subsequent K(+) stimulus. The effect size of glucose increased with physiological temperature whereas that of high K(+) did not. The mobility in the x/y-dimension and the fusion rates were little affected by the stimuli, in contrast to secretion. Fusion and secretion, however, had the same temperature dependence. Granules that appeared and fused within one image sequence had significantly larger caging diameters than pre-existent granules that underwent fusion. These in turn had a different mobility than residence-matched non-fusing granules. In conclusion, delivery to the membrane, tethering and fusion of granules are differently affected by insulinotropic stimuli. Fusion rates and secretion do not appear to be tightly coupled.

Observer-independent quantification of insulin granule exocytosis and pre-exocytotic mobility by TIRF microscopy.

Total internal reflection fluorescence microscopy of fluorescently labeled secretory granules permits monitoring of exocytosis and the preceding granule behavior in one experiment. While observer-dependent evaluation may be sufficient to quantify exocytosis, most of the other information contained in the video files cannot be accessed this way. The present program performs observer-independent detection of exocytosis and tracking of the entire submembrane population of insulin granules. A precondition is the exact localization of the peak of the granule fluorescence. Tracking is based on the peak base radius, peak intensity, and the precrossing itineraries. Robustness of the tracking was shown by simulated tracks of original granule patterns. Mobility in the X-Y dimension is described by the caging diameter which in contrast to the widely used mean square displacement has an inherent time resolution. Observer-independent detection of exocytosis in MIN6 cells labeled with insulin-EGFP is based on the maximal decrease in fluorescence intensity and position of the centroid of the dissipating cloud of released material. Combining the quantification of KCl-induced insulin exocytosis with the analysis of prefusion mobility showed that during the last 3 s pre-exocytotic granules had a smaller caging diameter than control granules and that it increased significantly immediately before fusion.

Bidirectional insulin granule turnover in the submembrane space during K(+) depolarization-induced secretion.

Like primary mouse islets, MIN6 pseudoislets responded to the depolarization by 40 mm KCl and the resulting increase in the free cytosolic Ca(2+) concentration ([Ca(2+) ](i) ) with a massive increase in insulin secretion, whereas 15 mm KCl had little effect in spite of a clear increase in [Ca(2+) ](i) . Analysis of insulin-enhanced green fluorescent protein (EGFP)-labeled granules in MIN6 cells by total internal reflection fluorescence (TIRF) microscopy showed that 40 mm KCl increased the number of short-term resident granules (<1 second presence in the submembrane space), while the total granule number and the number of long-term resident granules decreased. The rates of granule arrival at and departure from the submembrane space changed in parallel and were two orders of magnitude higher than the release rates, suggesting a back-and-forth movement of the granules as the primary determinant of the submembrane granule number. The effect of 15 mm KCl resembled that of 40 mm but did not achieve significance. Both 15 and 40 mm KCl evoked a [Ca(2+) ](i) increase, which was antagonized by 10 µm nifedipine. Nifedipine also antagonized the effect on secretion and on granule number and mobility. In conclusion, during KCl depolarization L-type Ca(2+) channels seem to regulate two processes, insulin granule turnover in the submembrane space and granule exocytosis.

Comparison of established and novel purity tests for the quality control of heparin by means of a set of 177 heparin samples.

The widespread occurrence of heparin contaminated with oversulfated chrondroitin sulfate (OSCS) in 2008 initiated a comprehensive revision process of the Pharmacopoeial heparin monographs and stimulated research in analytical techniques for the quality control of heparin. Here, a set of 177 heparin samples from the market in 2008 as well as pure heparin sodium spiked with defined amounts of OSCS and DS were used to evaluate established and novel methods for the quality control of heparin. Besides (1)H nuclear magnetic resonance spectroscopy (NMR), the assessment included two further spectroscopic methods, i.e., attenuated total reflection-infrared spectroscopy (ATR-IR) and Raman spectroscopy, three coagulation assays, i.e., activated partial thromboplastin time (aPTT) performed with both sheep and human plasma and the prothrombin time (PT), and finally two novel purity assays, each consisting of an incubation step with heparinase I followed by either a fluorescence measurement (Inc-PolyH-assay) or by a chromogenic aXa-assay (Inc-aXa-assay). NMR was shown to allow not only sensitive detection, but also quantification of OSCS by using the peak-height method and a response factor determined by calibration. Chemometric evaluation of the NMR, ATR-IR, and Raman spectra by statistical classification techniques turned out to be best with NMR spectra concerning the detection of OSCS. The validity of the aPTT, the current EP assay, could be considerably improved by replacing the sheep plasma by human plasma. In this way, most of the contaminated heparin samples did not meet the novel potency limit of 180 IU/mg. However, also more than 50% of the uncontaminated samples had <180 IU/MG. In contrast to the aPTT, the PT specifically detects OSCS and other heparin mimetics (LOD 3%). About ten times more sensitive are both the Inc-PolyH-assay and the Inc-aXa-assay, two rapid and simple quantification assays for heparin mimetics. The determined OSCS contents of the heparin samples excellently correlated with those calculated from the NMR spectra. In conclusion, NMR proved to be the current spectroscopic method of choice. The two two-step-assays represent options to supplement NMR, especially as tests for the initial screening, since they detect any heparin mimetic without requiring special expertise for interpretation of the results.

Composition of OSCS-contaminated heparin occurring in 2008 in batches on the German market.

In 2008, some 900 cases of adverse events associated with the use of heparin were reported to the Food and Drug Administration of USA and the Federal Institute of Drugs and Medical Devices in Germany. 238 patients died from heparin in the USA. In March 2008, oversulfated chondroitin sulfate (OSCS) was identified to be responsible for these cases. NMR spectroscopic evaluation of heparin samples revealed OSCS, dermatan sulfate (DS), chondroitin sulfate A and C as well as various residual solvents to be present in heparin batches, which could not be identified by means of conventional methods described in various pharmacopoeias at that time. In order to evaluate the situation on the German market, 145 representative samples were collected in 2008 and analyzed by means of 1H NMR spectroscopy, water determination, optical rotation and sheep plasma clotting assay. 66 samples were found to contain pure heparin, 51 samples heparin plus DS, 5 samples heparin plus OSCS, and 23 samples heparin, DS and OSCS, each in varying amounts. In 94 out of 145 batches especially ethanol was found in strongly varying amounts up to about 9.5%. Traces of acetone and formic acid were found with concentrations up to 0.04%, as well as sodium acetate and methanol up to 0.5%. Additionally, in many batches the content of water was found to be relatively high. Whereas the optical rotation was able to identify samples with a high contamination of OCSC, all samples tested fulfilled the requirements of the anticoagulation potency assay of the European Pharmacopoeia 6.0. The presented analysis of a representative set of heparin samples proves the suitability of 1H NMR spectroscopy for the quality control of heparin of both glycosaminoglycans and residual solvents.

A small-molecule inhibitor of Nipah virus envelope protein-mediated membrane fusion.

Nipah virus (NiV), a highly pathogenic paramyxovirus, causes respiratory disease in pigs and severe febrile encephalitis in humans with high mortality rates. On the basis of the structural similarity of viral fusion (F) proteins within the family Paramyxoviridae, we designed and tested 18 quinolone derivatives in a NiV and measles virus (MV) envelope protein-based fusion assay beside evaluation of cytotoxicity. We found five compounds successfully inhibiting NiV envelope protein-induced cell fusion. The most active molecules (19 and 20), which also inhibit the syncytium formation induced by infectious NiV and show a low cytotoxicity in Vero cells, represent a promising lead quinolone-type compound structure. Molecular modeling indicated that compound 19 fits well into a particular protein cavity present on the NiV F protein that is important for the fusion process.