In vivo dopamine receptor binding studies with a non-radioactively labeled agonist,dipropyl-5, 6-ADTN

Evidence is presented that a low dose of peripherally administered N, N-dipropylamino-5, 6-dihydroxytetralin (DiPr-5, 6-ADTN) specifically labels dopamine (DA) receptors in rat brain.Concentrations of this potent DA receptor agonist were determined by a highly selective method using reversed phase liquid chromatography and amperometric detection. The binding characteristics satisfy all criteria regarding saturability, stereospecificity, regional distribution and relation with pharmacological effects that are associated with DA receptor interactions. A rough estimation of the density of binding sites in the striatum resulted in values of 60–70 pmol/g. Lesioning the nigrostriatal pathway does not significantly alter the amount of ligand bound, nor do pretreatments with serotonergic, α-adrenergic or β-adrenergic antagonists. DiPr-5, 6-ADTN has thus been shown to be a useful ligand for labeling central DA receptors and a powerful tool in the study of DA-ergic mechanisms.     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

Alterations in copper and collagen metabolism in the Menkes syndrome and a new subtype of the Ehlers-Danlos syndrome

Cultured fibroblasts of 13 patients with the Menkes syndrome and two with a new subtype (type IX) of the Ehlers-Danlos syndrome (E-D IX patients) showed many very similar abnormalities in their copper and collagen metabolism. Both cell types had markedly increased copper concentrations and 64Cu incorporation, and this cation accumulated in metallothionein or a metallothionein-like protein, as previously established for Menkes cells. Histochemical staining indicated that copper was distributed diffusely throughout the cytoplasm in both cell types, this location being consistent with the accumulation in metallothionein. Both fibroblast types also had markedly low lysyl oxidase activity and distinctly increased extractability of newly synthesized collagen, whereas no abnormalities were present in cell viability, duplication rate, prolyl 4-hydroxylase activity, or collagen synthesis rate. A high negative correlation (P less than 0.001) was found in the pooled group of Menkes and E-D IX cells between cellular copper concentration (r = 0.804) or 64Cu incorporation (r = 0.863) and the logarithm of lysyl oxidase activity. There was also a high positive correlation (P less than 0.001) between cellular copper concentration and incorporation (r = 0.869). One of the two E-D IX patients was also shown to have similar changes in lysyl oxidase activity and collagen extractability in the skin biopsy specimen, suggesting that the abnormalities observed in cultured cells are similar to those present in vivo. The only distinct abnormality found in the cells of the parents of the E-D IX patients was an increased 64Cu incorporation in those of the mother, this finding being consistent with X-linked inheritance of the disorder.     

Nucleotide pools of growing,synchronized and stressed cultures of Saccharomyces cerevisiae

High pressure liquid chromatography has been used to study the acid soluble nucleotide pool of Saccharomyces cerevisiae under different conditions of growth. ATP, ADP, AMP, NAD, GTP, UTP, UDP, CTP, CDP, and UDP-sugars plus UMP could be separated and were found in concentrations higher than 0.1 mumol per g yeast cell dry weight (= detection limit). During glucose-limited continuous culture the levels of individual nucleotides depended on the growth rate, which was most pronounced with pyrimidine (uridine, cytidine) nucleotides. The energy charge (E.C.) remained high (0.9) at all growth rates (0.07-0.3 h-1). During synchronized growth at a constant growth rate (0.11 h-1) almost all nucleotide levels and the E.C. remained at constant values with the only exception of UDP-sugars and UMP of which increased levels were found during the phase of budding. Under conditions of metabolic stress (addition of antimycin A, deoxyglucose plus iodoacetate) pronounced changes in the levels of purine (adenine and guanine) nucleotides and the E.C. were observed. All other nucleotides were less influenced by these conditions. Only under these conditions IMP accumulation was observed. The results strongly argue against the significance of purine nucleotide or E.C. measurements under viable conditions. In contrast, changes in the levels of pyrimidine nucleotides seem to be indicative of changes in the flux through the metabolic pathways where they act as coenzymes.     

Effects of potassium,lanthanum and black widow spider venom on miniature synaptic potentials in theTorpedo electroplax

Summary Using a method of focal drug application it is demonstrated that high potassium concentration, lanthanum, and black widow spider venom accelerate spontaneous transmitter release inTorpedo electric tissue.This investigation was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 138). Thanks are due to Dr. R. Martin and the staff of the Stazione Zoologica, Naples, for supplyingTorpedo, and Dr. N. Frontali, Rome, for a gift of frozen black widow spiders.     

The effect of energization on the apparent Michaelis–Menten constant for oxyge in nmitochondrial respiration

Lineweaver-Burk plots of 1/v against 1/[O(2)] for rat liver mitochondrial respiration with succinate or ascorbate+NNN'N'-tetramethyl-p-phenylenediamine as substrates are non-linear. In state 3u (uncoupled by trifluoromethoxycarbonyl cyanide phenylhydrazone) such plots tend to be concave upward, whereas in state 4 (energized) the plots were concave downward. The apparent K(m) for oxygen is larger in state 4 than in state 3u, despite the higher turnover in the latter system. It is postulated that at least one reversible reaction occurs between cytochrome c and cytochrome c oxidase, whose rate is increased on energization (reversed electron transfer); a model including such a reaction is proposed which accounts semiquantitatively for the observations.     

Effect of arterial perfusion on net water flux and active sodium transport across the isolated skin ofBufo bufo bufo (L.)

Summary A method has been developed which allows perfusion of the blood vessels in isolated pelvic skins ofB. bufo. The effect of various doses of vasotocin (AVT) on net water flux (inside medium 220 mOsM, outside medium 11 mOsM) and active sodium transport were compared in perfused and unperfused skins. The unstimulated water flux (Fig. 3) and the active sodium transport (Fig. 6) were unaffected by perfusion. The threshold for stimulation of water flux was between 0.01 and 0.1 nM vasotocin in perfused skins and between 0.1 and 1 nM in unperfused skins. The threshold for stimulation of active sodium transport was between 0.005 and 0.05 nM vasotocin in perfused skins and between 0.05 and 0.5 nM in unperfused skins. The maximal water flux through perfused skins, 4 l/cm²min, was obtained at 1 nM vasotocin. At 10 nM vasotocin the water flux was only 0.7 l/cm²min in unperfused skins. The maximal active sodium transport was approximately of the same magnitude in perfused and unperfused skins, at 0.5 mM and at 50 nM, respectively.