Individual organelle pH determinations of magnetically enriched endocytic organelles via laser-induced fluorescence detection

The analysis of biotransformations that occur in lysosomes and other endocytic organelles is critical to studies on intracellular degradation, nutrient recycling, and lysosomal storage disorders. Such analyses require bioactive organelle preparations that are devoid of other contaminating organelles. Commonly used differential centrifugation techniques produce impure fractions and may not be compatible with microscale separation platforms. Density gradient centrifugation procedures reduce the level of impurities but may compromise bioactivity. Here we report on simple magnetic setup and a procedure that produce highly enriched bioactive organelles based on their magnetic capture as they traveled through open tubes. Following capture, in-line laser-induced fluorecence detection (LIF) determined for the first time the pH of each magnetically retained individual endocytic organelle. Unlike bulk measurements, this method was suitable to describe the distributions of pH values in endocytic organelles from L6 rat myoblasts treated with dextran-coated iron oxide nanoparticles (for magnetic retention) and fluorescein/TMRM-conjugated dextran (for pH measurements by LIF). Their individual pH values ranged from 4 to 6, which is typical of bioactive endocytic organelles. These analytical procedures are of high relevance to evaluate lysosomal-related degradation pathways in aging, storage disorders, and drug development.     

Kinetics of thiuram–accelerated sulfur vulcanization

On the basis of continuous measurements in a Vuremo curemeter at 145°C, kinetics of tetramethylthiuram disulfide (TMTD)-accelerated sulfur vulcanization of natural rubber has been investigated. It was found that the cure rates increase with increasing TMTD concentration, the sulfur content being kept constant, up to a TMTD:S weight ratio of 2:1. Beyond this value, the cure rates again decrease. This TMTD:S ratio corresponds to 3.8 gram atoms of sulfur per mole TMTD, and it is in good agreement with findings that in TMTD-accelerated sulfur vulcanization systems the peak value of zinc dimethyldithiocarbamate (ZnDMDC) formation reaches an endvalue when the stocks contain 4 gram atoms of sulfur per mole TMTD. These facts lead us to suppose that ZnDMDC is the actual accelerator in TMTD-accelerated sulfur systems. Support for this view derives from our experiments with model curing systems as well as from the generally known practical experience that dithiocarbamates are faster accelerators than thiuram disulfides. For the reasons described above and for the finding that the dependences of the ultimate extent of vulcanization (network chain density) on the concentration of TMTD in the absence and in the presence of elemental sulfur are analogous, the mechanism of thiuram-accelerated sulfur vulcanization is very probably similar to that of sulfur-free thiuram vulcanization.     

Efficiency of metal activators of accelerated sulfur vulcanization

The effects of copper, mercury, nickel, zinc, cadmium, indium, magnesium, and calcium stearates on the course of N-cyclohexyl-2-benzthiazylsulphenamide-accelerated sulfur vulcanization of natural rubber have been investigated on the basis of curemeter measurements at 145°C. The differences in the efficiencies of these metal activators of accelerated sulfur vulcanization have been discussed from the points of view of the electron configurations of the metals and their affinities to sulfur. We attempted to determine why zinc oxide is generally accepted as the best metal vulcanization activator. © 1993 John Wiley & Sons, Inc.     

Kinetics of sulfur-free thiuram vulcanization

Sulfur-free thiuram vulcanization has been investigated at temperatures from 160° to 190°C over 0.5 to 600 min. Continuous measurements in a VUREMO curemeter were used to estimate the extent of crosslinking, which was plotted against cure time. Simultaneously the values of the network chain density were calculated from swelling measurements on the vulcanizates. The cure curves show clearly an induction period (t_i), then fast crosslinking, a partial degradation, a “long-time” crosslinking, and finally a slow, limited degradation. Apart from the induction period, the kinetic graphs are satisfactory represented by a rate equation assuming three independent first-order reactions: fast crosslinking, degradation, and slow crosslinking. The rate equation contains seven kinetic parameters. Over the temperature range studied, there is no difference between the values of activation energy for fast crosslinking, for degradation, for slow crosslinking, and for t. Due to the presence of thiourea, the values of the induction period, the rate constant, and the extent of slow crosslinking are decreased. Simultaneously the activation energies calculated from degradation and slow crosslinking are significantly increased. On the basis of the above results, the mechanism of the sulfur-free thiuram vulcanization, in which ionic and radical reactions take place, is discussed.     

Effect of aerosil on the course of thiuram accelerated sulfur vulcanization

Tetramethylthiuram disulfide (TMTD)-accelerated sulfur vulcanization of natural rubber has been investigated at temperatures from 100°C to 145°C. Continuous measurements in a Vuremo curemeter were used to estimate the extent of crosslinking, which was plotted against cure time. The cure curves as well as their linearized forms (dependences of the logarithm of the extent of vulcanization on the cure time) clearly show that at lower cure temperatures the course of the vulcanization differs significantly from the first-order rate law. These digressions have been removed by the addition of a highly dispersed silica gel, Aerosil, which simultaneously speeds up the course of the vulcanization up to the value corresponding to the rate of zinc dimethyldithiocarbamate (ZnDMDC)-accelerated sulfur vulcanization. These results are in accordance with our recent theory supposing that ZnDMDC is the actual accelerator in TMTD-accelerated sulfur systems. In the presence of Aerosil, the formation of ZnDMDC from TMTD is catalyzed via dispersed silica gel. Support for this view derives from the temperature dependences of vulcanization reactions. The activation energies of TMTD-accelerated sulfur vulcanizations in the absence (31 kcal/mole) and in the presence of Aerosil (23.5 kcal/mole) correspond exactly to the values calculated from the rate constants of the thiuram decrease in TMTD-accelerated vulcanization (30 kcal/mole) and from the rate constants of crosslinking in the dithiocarbamate-accelerated sulfur vulcanization (23 kcal/mole), respectively.     

Die wirtschaftlichen Transformationsgesetze und Transformatorenentwürfe bei Berücksichtigung der wahren Änderung der Eisenverluste mit der magnetischen Liniendichte

Zusammenfassung Bei der Berechnung des Transformatorenentwurfes wird die wahre Änderung der Eisenverluste mit der Liniendichte berücksichtigt. Dadurch werden im allgemeinen niedrigere optimale Werte der Liniendichte gewonnen als nach dem bisher allgemein angewandten quadratischen Näherungsgesetz. Durch Interpretation der die Kleinstwerte bestimmenden Gleichungen werden wirtschaftliche Transformationsgesetze abgeleitet, deren Fassung zum Teil neu ist, zum Teil Modifikationen schon bekannter Gesetze darstellt, die durch die Einführung der wahren Änderung der Eisenverluste mit der Liniendichte bedingt sind. Ein Berechnungsbeispiel ergänzt die Ausführungen.Zusammenstellung der verwendeten Formelzeichen a Spezifische Jahreskosten der Eisenverluste in RM/kgG^u - b spezifische Jahreskosten der Stromwärme in RM · mm⁴/kg · A² - B Säulenliniendichte in G - B _o Ausgangsliniendichte in G - C 0,25··_e·f_e·10^–3 - D Kernkreisdurchmesser in Zentimeter - f Frequenz - f _e Eisenfüllfaktor, bezogen auf den Kernkreis - f _w Fensterfüllfaktor, gemessen von Kernkreis zu Kernkreis und von Jochkante zu Jochkante - G _e Kerneisengewicht in Kilogramm - G _w Wicklungsgewicht in Kilogramm - h Verhältnis der Material- und Verlustkosten der Joche - h _e jährliche Betriebsstunden der Eisenwärme - h _w jährliche Betriebsstunden der Stromwärme - i Stromdichte in A/mm² bei Vollast - k Jochverstärkungsverhältnis - K jährliche Transformationskosten in RM - K _Ve jährliche Eisenverlustkosten, d. h. jährliche Kosten der Eisenwärme und der für sie notwendigen Kühleinrichtung in RM - K _Vw jährliche Stromwärmekosten, d. h. jährliche Kosten der Stromwärme und der für sie notwendigen Kühleinrichtung in RM - L Lagrangesche Hilfsfunktion - n _w·f_w/ _e·f_e - N Transformatorscheinleistung in kVA - P _e spezifische Jahreskosten für das Kerneisen, d. h. die auf ein Kilogramm Kerneisen entfallenden jährlichen Kapitalskosten in RM/kg - P _k spezifische Jahreskosten für die Kühleinrichtung, d. h. jährliche Kapitalskosten der auf ein kW Verluste entfallenden Kühleinrichtung in RM/kW - P _Ve Preis der kWh für die Eisenwärme in RM/kWh - P _Vw Preis der kWh für die Stromwärme in RM/kWh - P _w spezifische Jahreskosten für das Wicklungsmaterial, d. h. die auf ein Kilogramm Wicklungsmaterial entfallenden jährlichen Kapitalskosten in RM/kg - t Volumen des über den Säulen liegenden Jocheisens/Jochquerschnitt × Kernkreisdurchmesser - u Exponent der die Abhängigkeit der Eisenverluste von der magnetischen Liniendichte darstellenden Näherungsfunktion - v ₀ Eisenwärmeverlust in W/kg bei der Ausgangsliniendichte B₀ - v _w Stromwärmeverlust in W/kg bei der Stromdichtei= 1 A/mm² - V _e Kerneisenverlustleistung in kW - V _s Säuleneisenverlustleistung in kW - x relative Säulenhohe =Säulenhöhe/Kernkreisdurchmesser - y relative Fensterbreite = Fensterbreite/Kernkreisdurchmesser - _e Wichte des Eisens in kg/dm³ - _w Wichte der Wicklung in kg/dm³ - Lagrangescher Hilfsfaktor