Immunofluorescence staining of thin-filament sections not participating in actomyosin crossbridges: Studies by use of a monoclonal antibody specific to actin

Summary Monoclonal antibodies (mcab) were produced in vitro by fusing mouse X63-Ag8.653 plasmacytoma cells with spleen cells from a Balb/c mouse immunized with primary cultures of chick skeletal muscle (pmcc). After cloning on agar, stable clones were obtained, the antibodies of which stain specifically the I-band of myofibrils in the immunofluorescence (IF) procedure. For further characterization of these mcab their affinities to muscle proteins were tested by immunoblotting and by enzyme-linked immunosorbent assay (ELISA). Mcab specific for actin were revealed by these criteria. One of the anti-actin antibodies, mcab 647, reveals a variety of IF-staining patterns on myofibrils. On rest-length myofibrils the I-band is labeled only. However, at sarcomere lengths below 2 m, where the thin filaments meet in the middle of the A-band and form a region of double overlap, an additional fluorescent band appears in this position. The fluorescence intensity of this band is increased significantly in shorter sarcomeres. Finally, when the I-band has disappeared at a sarcomere length of 1.5 m, fluorescence is located exclusively in the middle of the A-band. These IF-staining patterns suggest that only those sections of the thin filament are stained that do not participate in actomyosin crossbridges.     

CFU-gm assay, cytochemical and electron microscopic studies in agar in patients with preleukemic syndrome and aplastic anemia

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.     

Chronic administration of an organophosphorus insecticide to rats alters cholinergic muscarinic receptors in the pancreas

Male rats were treated for 10 days with the organophosphorus insecticide, acetylcholinesterase inhibitor, O,O-diethyl S-[2-(ethylthio)ethyl]phosphorodithioate (disulfoton, 2 mg/kg/day by gavage). At the end of the treatment, binding of [³H]quinuclidinyl benzilate ([³H]QNB) to cholinergic muscarinic receptors and cholinesterase (ChE) activity were assayed in the pancreas. Functional activity of pancreatic muscarinic receptor was investigated by determining carbachol-stimulated secretion of α-amylase in vitro. ChE activity and [³H]QNB binding were significantly decreased in the pancreas from disulfoton-treated rats. The alteration of [³H]QNB binding was due to a decrease in muscarinic receptor density with no change in the affinity. Basal secretion of amylase from pancreas in vitro was not altered, but carbachol-stimulated secretion was decreased. The effect appeared to be specific since pancreozymin was able to induce the same amylase release from pancreases of control and treated rats. The results suggest that repeated exposures to sublethal doses of an organophosphorus insecticide lead to a biochemical and functional alteration of cholinergic muscarinic receptors in the pancreas.     

Temporal Distribution of [3H]-Imipramine Binding in Rat Brain Regions is Not Changed by Chronic Methamphetamine

Specific binding of [³H]-imipramine in the rat suprachiasmatic nuclei, occipital cortex and caudate putamen underwent significant and replicable changes throughout 24 hr under a light-dark cycle or under constant conditions. Daily variations were also found in the medial and dorsal raphe nuclei and the lateral hypothalamus. Methamphetamine, a psychoactive drug with marked effect on circadian rhythms in physiological and hormonal parameters and adrenergic receptors, did not have any significant effect on imipramine binding rhythms in eight discrete brain regions. Thus a drug known to reduce serotoninergic neurotransmission did not change characteristics of the modulatory binding site related to serotonin uptake.     

Apolipoprotein A-I isoforms in human lymph: effect of fat absorption

The effect of fat feeding (100 g of cream) on the apoA-I isoproteins distribution has been analyzed by two-dimensional gel electrophoresis in the chylomicrons, VLDL, LDL, and HDL isolated from the thoracic duct lymph of patients undergoing lymph drainage for immunosuppression, Isoforms apoA-I3 and apoA-I4 are the most abundant apoA-I isoproteins in plasma lipoproteins as well as in lymph lipoproteins collected in the fasting state. Fat feeding, on the other hand, results in a marked change in the apoA-I isoform pattern in lymph chylomicrons and VLDL, with a significant increase in the relative concentration of the apoA-I1 isoform. As a result the total concentration of this isoprotein in the lymph increased. The data indicate that fat feeding is associated with major changes in the distribution of the apoA-I isoforms in the lymph (d less than 1.006 g/ml lipoproteins), which may be of significance in their plasma catabolism.     

Preparative Electrophoresis of Isotopically Labeled L-Cell Interferons

The preparative method of polyacrylamide gel electrophoresis was adapted for purification and characterization of isotopically labeled L-cell interferons. Re-covery of interferon activity was quantitative, and purification and resolution were comparable to those obtained by analytical polyacrylamide gel electrophoresis. Ultimate specific activities attainable ranged from 2 x 10(6) to 3 x 10(6) international units per mg of protein.     

Der Einfluß von Elektrolyten auf Chloroplasten beim Gefrieren und Trocknen

Summary The effect of freezing, desiccation and various electrolytes on photophosphorylation, electron transport and some enzyme reactions of isolated spinach chloroplasts has been investigated. Freezing of broken chloroplasts took place at-25°C for 3 hrs; desiccation was performed at +2°C in vacuo over CaCl₂ for 3 hrs. The influence of various electrolytes during freezing or drying or during incubation of thylakoids or stroma enzymes for 3 hrs at +2°C in electrolyte solutions was determined. After treatment, the activities of a number of enzymes and enzyme systems were measured under normal conditions, e. g. in the absence of elevated electrolyte levels in a reaction medium which contained only the substrates and cofactors which are necessary for the respective enzyme reactions.Only photophosphorylation and electron transport were affected by freezing, desiccation and high concentrations of electrolytes; various soluble enzymes investigated here were not inactivated under the same conditions. In general, mild dehydration and lower concentrations of electrolytes resulted in an irreversible inactivation of ATP synthesis but did not impair ferricyanide reduction. With increasing dehydration or at higher concentrations of electrolytes the Hill reaction was also inhibited. In a certain range of dehydration and electrolyte concentration uncoupling of photophosphorylation from electron transport took place. Sugar protects the sensitive structures against the deleterious effect of both dehydration and high concentration of electrolytes.Various electrolytes affected thylakoid membranes differently. Inactivation of the membranes increased with increasing ion radius and decreasing hydration envelope of univalent or divalent cations. Divalent cations were more destructive than univalent cations. Anions did not follow these rules. Within a group of similar anions (halides or organic anions) effectivity decreased with increasing hydration envelope. On a molar basis, polyvalent anions were less effective than univalent anions. Inactivation by anions followed Hofmeister's series in seversed order. However, exceptions were observed and it appears that various ions affect the membrane in a specific manner.Inactivation of photophosphorylation and electron transport due to freezing or desiccation is identical to that due to high concentrations of electrolytes. This suggests that during dehydration due to freezing or drying the concentration of electrolytes in the remaining solution is responsible for the inactivation of the sensitive membranes.     

Flavonglykoside in Blüten vonPaeonia arborea undPaeonia suffruticosa

Summary Three glycosides have been isolated fromPaeonia arborea: kaempferol-3--glucoside-7--glucoside (Paeonoside), apigenin-7--glucoside, and apigenin-7-rhamnoglucoside (Rhoifolin).Paeonia suffruticosa also contains these three compounds but its main glycoside is kaempferol-3--glucoside (astragalin), which is present inPaeonia arborea only in traces.