The amount and nature of glutathione transferases in rat liver microsomes determined by immunochemical methods

The amount and nature of glutathione transferases in rat liver microsomes were determined using immunological techniques. It was shown that cytosolic glutathione transferase subunits A plus C, and B plus L were present at levels of 2.4 ± 0.6 and 1.5 ± 0.1 μg/mg microsomal protein, respectively. These levels are 10-times higher than those for non-specific binding of cytosolic components judging from the distribution of lactate dehydrogenase, a cytosolic marker. The possibility that a portion of these glutathione transferases is functionally localized on the endoplasmic reticulum is discussed. A previously described microsomal glutathione transferase which is distinct from the cytosolic enzymes is present in an amount of 31 ± 6 μg/mg microsomal protein.     

Das Problem der Spezifizität des Sabin-Feldman Farbtestes

Zusammenfassung Bei der Diagnostik der Toxoplasmosis, wie auch bei epidemiologischen Studien mit Hilfe dieses Testes, st?szt man auf die Nicht-Reproduzierbarkeit in engen Grenzen und auf die Frage welcher Wert niedrigen Serumtitern beigelegt werden soll. Die Technik des Sabin-Feldman Farbtestes ist vonvan Soestbergen studiert und verbessert worden, wodurch der Test auf diese Weise reproduzierbar geworden ist. Mit Hilfe dieser Technik und experimentell ist FrauMas Bakal besch?ftigt Probleme der Aspezifizit?t zu beantworten. Bis jetzt k?nnen keine Motive angeführt werden die gegen die Spezifizit?t des Testes sprechen. Vortrag auf der I Tagung der tschechoslowakischen Parasitologen in Prag 6–10 Oktober 1957.     

Postmortem Degradation Alters Fluorographic Labeling Patterns and Affinities of Benzodiazepine Binding Proteins

To investigate the effect of endogenous proteolysis on the molecular weights of the benzodiazepine binding proteins, brains of trout, chicken, and rat were removed immediately after death and stored at room temperature for various periods of time before they were frozen. Photoaffinity labeling of membranes with [3H]flunitrazepam, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, revealed proteolytic fragments of 47K in trout, chicken, and rat. The proteolysis set in rapidly after death. Seemingly in parallel with the degradation observed fluorographically, the affinity for [3H]flunitrazepam increased without systematic changes in receptor density. The degradation pattern was not identical to that of the photolabeled trypsinized benzodiazepine binding proteins. The endogenous proteolytic fragments were deglycosylated in two steps. In conclusion, proteolytic effects must be taken into account when interpreting labeling patterns and binding parameters.     

Model system using coliphage phi X174 for testing virus removal by air filters.

Short-term (15-min-duration) and long-term (5- to 6-day-duration) test procedures have been developed for determining the efficiency of the removal of bacteriophage phi X174 by air-sterilizing filters. These procedures were sensitive enough to measure a 10(8)-fold reduction in the number of bacteriophage. A filter commonly used in industrial air sterilizations (Domnick-Hunter Bio-X borosilicate glass) effected a 10(8)-fold removal of viable phage in both short-term and long-term tests. A prototype low-flux, hollow-fiber membrane gave similar results; however, a prototype high-flux, hollow-fiber membrane removed only about 99.999% of the bacteriophage in short-term tests.     

Elicitation of volatile compounds in photomixotrophic cell culture of Petroselinum crispum

Photomixotrophic cells of Petroselinum crispum accumulated >500 mg chlorophyll per kg wet weight and grew well in a broad range of phytoeffector conditions. Autoclaved fungal cells were lethal for photoheterotrophic cells, but induced in photomixotrophic cells the formation of volatile n-alkanes, phthalides, coumarins, and elemicine. Most of the compounds elicited reached a concentration maximum between 20 and 30 h after addition of the mycelium, whereas the group of n-alkanes increased steadily during the 90 h monitored. Maximum concentrations were: 12 mg of graveolone, 1 mg of bergapten, 0.5 mg of sedanenolide, and 0.5 mg of n-tetradecane per 1 nutrient medium. A dose/effect relationship was found; 10 to 25 g of fungal wet weight per 1 culture medium resulted in maximum accumulation of volatiles. The formation of volatiles by photomixotrophic in vitro cells is discussed as an integral part of plant responses to ecological stress.     

Further indications of the multicomponent nature of the acrosome reaction-inducing substance of human follicular fluid

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran-coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR-inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with ³H-progesterone-labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR-inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction-inducing substance (ARIS) appears to be a complex of progesterone and a progesterone-binding protein, which was shown to be identical with the plasma protein corticosteroid-binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley-Liss, Inc.     

Redox-state of intactNitella cells: dependency on intracellular pH and photosynthesis

Summary The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (E_RED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, E_RED values of+237±93mVand+419±51 mV with respect to a normal H⁺-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state E_RED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of E_RED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic E_RED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic E_RED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10M 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), E_RED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.Abbreviations E_M transmembrane voltage - E_RED redox-state voltage - E₀ midpoint-redox-voltage - APW artificial pond water - CEF cytoplasm enriched fragment     

Low temperature cultivation — A step towards process optimisation

Adherent recombinant BHK cells were cultivated at temperatures between 30 and 37°C. Batch and repeated-batch-cultivations in a 2-litre bioreactor showed a significant influence on metabolism and cell growth. The low-temperature-cultivations showed a lower growth rate and a lower glucose consumption rate and, therefore, less lactate production. On the other hand, the maximum cell density and productivity seemed not to be affected by the temperature reduction.