Distribution of carotenoids in sub-cellular and sub-plastidic fractions of radish seedlings (Raphanus sativus) grown in the presence of bleaching herbicides

The intracellular and intraplastidic distribution of carotenoids has been investigated in radish seedlings grown in the presence of the herbicides amitrole and SAN 6706. Both herbicides caused bleaching and the plants became deficient in chlorophylls and the usual chloroplast cyclic carotenoids, but accumulated the acyclic carotenoid biosynthetic intermediates 15-cis-phytoene and all-trans-lycopene. In both the untreated and herbicide-treated plants all carotenoids, including phytoene and lycopene, were contained in the plastid. In all cases the normal cyclic carotenoids were located virtually exclusively in the thylakoid or prothylakoid fraction. In amitrole-treated plants, lycopene also was contained only in the thylakoid fraction, whereas phytoene, in these and in SAN 6706-treated plants, was detected in both the thylakoid fraction and an envelope preparation. Possible implications for the biosynthesis of the carotenoids are discussed.     

Incorporation von [D-4,5-3H]-Ieucine und [2-14C]-Acetat in die photosynthetischen Pigmente und Chinone von Chlorella pyrenoidosa

Der Metabolismus der photosynthetischen Pigmente und Chinone von Chlorella wurde mit Hilfe von ¹⁴CO₂, [2-¹⁴C]-Acetat und [D-4,5-³H]-Leucin als Vorstufen der Isoprenoidsynthese untersucht. Auch wurde untersucht, ob Leucin als Vorstufe in der Biosynthese der Terpenoide fungiert. Die Verwendung der Doppelmarkierung sollte bestehende Unterschiede in der Regulation und Biosynthese der Isoprenoide der Chloroplasten, Mitochondrien und des Zytoplasmas aufdecken. Neben der Verwendung von Radioisotopen wurde die Inkorporation von Deuterium in die Carotinoide verwendet, um den turnover der Prenyl-Lipide direkt nachzuweisen. In tracer-kinetischen Untersuchungen mit ¹⁴CO₂ konnte gezeigt werden, daß ¹⁴-C-α-Carotin zu ¹⁴C-Lutein und ¹⁴C-ß-Carotin zu ¹⁴C-Zeaxanthin umgesetzt wird. Eine Vorstufenfunktion läßt sich auch für Plastohydrochinon-9 nachweisen, das in Plastochinon-9 umgesetzt wird. Die Markierungskinetik der einzelnen Prenyl-Lipide deutet auf einen schnellen turnover hin. Anhand der ¹⁴C-Inkorporationskinetik wurden Halbwertszeiten im Bereich von 30 min bis 220 min ermittelt, während sich aus den Dekorporationskinetiken Zeiten von 9 bis 113 h ergeben. Prenyl-Lipide wie die Carotine, Chlorophyll a und Plastochinon-9, die einerseits Vorstufenfunktion besitzen, andererseits aber auch direkt an der Photosynthese beteiligt sind, werden schneller umgesetzt als ihre Folgeprodukte. Somit scheint ein direkter Zusammenhang zwischen der Funktion des Prenyl-Lipides im Chloroplasten und seinem Umsatz zu bestehen. Sowohl Acetat wie auch Leucin werden von Chlorella als Vorstufen in der Terpenoidbiosynthese verwendet. Acetat wird aber wesentlich schneller inkorporiert als Leucin, was vielleicht darauf zurückzuführen ist, daß Leucin im Zytoplasma zu Acetat oder Mevalonsäure metabolisiert wird und dann in den Chloroplasten gelangt. Auch im tracer-kinetischen Experiment mit ¹⁴C-Acetat und ³H-Leucin läßt sich eine Vorstufen-Produkt-Beziehung für die Chlorophylle, Carotinoide und Chinone zeigen. Im Vergleich zu den Experimenten mit ¹⁴CO₂ zeigen die tracer-kinetischen Experimente mit ¹⁴C-Acetat und ³H-Leucin, daß die Chloroplasten-Isoprenoide wesentlich langsamer metabolisiert werden und ihre Umsatzraten mehr jenen entsprechen, die aus den ¹⁴C-Dekorporationskinetiken des ¹⁴CO₂-Experimentes resultieren. Es zeigt sich aber auch hier, daß jene Prenyl-Lipide, die direkt an der Photosynthese beteiligt sind, schneller metabolisiert werden. Daß die Chloroplasten-Isoprenoide in autotroph kultivierten Chlorellen einem ständigen turnover unterliegen, läßt sich auch sehr eindrucksvoll mit Hilfe der Deuterium-Inkorporation für die Carotine zeigen. Die aus der Deuterium-Inkorporation erhaltenen Umsätze der Carotine entsprechen in etwa denen, die sich aus ihrer Dekorporationskinetik im tracer-kinetischen Experiment mit [2-¹⁴C]-Acetat und [D-4,5-³H]-Leucin ergeben, zeigen allerdings, daß a-Carotin wesentlich schneller umgesetzt wird als ß-Carotin. Die Untersuchungen wurden durch ein Stipendium der Deutschen Forschungsgemeinschaft im Austausch mit der British Royal Society an K.H.G. unterstützt, wofür wir uns bedanken.     

Untersuchungen zur Autonomie des Chloroplasten in bezug auf seine Terpenoidbiosynthese

Isolierte Spinatchloroplasten mit einer photosynthetischen Aktivität von 280 μmol Sauerstoff je mg Chlorophyll und h und einer Intaktheit von 85 % wurden mit¹⁴C-markiertem CO₂, Phosphoglycerat, Phosphoenolpyruvat, Pyruvat, Acetat und Mevalonat für 60 min inkubiert und die Inkorporation von Radioaktivität in die Chlorophylle und Carotinoide untersucht. Alle verwendeten Substrate wurden vom Chloroplasten aufgenommen und Radioaktivität in die Chlorophylle inkorporiert. Auch die Carotinoide nehmen mit Ausnahme von Phosphoenolpyruvat Radioaktivität von den einzelnen Substraten auf. Aus den Inkorporationsexperimenten wird geschlossen, daß Chloroplasten in bezug auf ihre Terpenoidbiosynthese autonom sind. Chloroplasten sind somit in der Lage, aus im Calvin-Zyklus fixiertem CO₂ über Phosphoglycerinsäure und Pyruvat Acetyl-Coenzym-A zu synthetisieren. Acetyl-Coenzym-A kann dann einerseits direkt zur Biosynthese von Fettsäuren verwendet werden, andererseits aber auch zur Biosynthese von Carotinoiden und den isoprenoiden Seitenketten der Chinone und Chlorophylle. Die Untersuchungen wurden durch eine Sachbeihilfe der Deutschen Forschungsgemeinschaft unterstützt.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

Acute N-methyl-D,L-aspartate administration stimulates the luteinizing hormone releasing hormone pulse generator in the ovine fetus

To assess whether fetal luteinizing hormone releasing hormone (LH-RH) neurosecretory neurons have the capacity to respond to an exogenous stimulus, a synthetic excitatory amino acid analogue, N-methyl-D-L-aspartate (NMDA; 15 mg/kg), was given rapidly intravenously to 8 chronically catheterized fetuses (130-142 days of gestation; term 147 +/- 3 days). All 8 fetuses exhibited a rise in plasma ovine luteinizing hormone (oLH) and ovine follicle-stimulating hormone (oFSH) within 5 min. The mean maximal increments of oLH (2.25 +/- 0.36 ng/ml) and oFSH (1.21 +/- 0.32 ng/ml) were significantly greater than in 6 normal saline-injected controls (oLH p < 0.0002; oFSH p < 0.03). The secretion of ovine prolactin (oPRL) and ovine growth hormone (oGH) was unaffected. LH-RH (5 microg) evoked a greater oLH response (p < 0.0009) and a greater oFSH response (p < 0.03) than NMDA (n = 6). Desensitization of the fetal gonadotrope by a potent LH-RH agonist, D-Trp6Pro9NEt-LH-RH (10 microg/day i.v. x 4 days), abolished the fetal oLH and the oFSH response to NMDA (n = 5). Moreover, D, L-2-amino-5-phosphonovalerate, a specific competitive antagonist for the NMDA receptor, completely inhibited the fetal oLH and oFSH response to NMDA, whereas D-L-2-amino-5-phosphonovalerate alone did not affect the plasma oLH or oFSH levels, the gonadotropin response to LH-RH, or the release of oGH or oPRL (n = 3). In primary ovine fetal pituitary cell cultures, NMDA (10(-10) to 10(-6) M) had no effect on oLH, oFSH, oGH, or oPRL secretion, whereas LH-RH stimulated oLH (10(-8) M; p < 0.0004) and oFSH (10(-8) M; p < 0. 0001) release, evidence that NMDA did not have a direct pituitary effect. The results suggest that NMDA induces oLH and oFSH secretion by stimulation of the fetal LH-RH pulse generator and is mediated by central NMDA receptors. Fetal LH and FSH secretion and the response to LH-RH decrease in late gestation in the ovine and human fetus. The relative importance of sex steroid dependent and sex steroid independent central nervous system inhibition in this developmental change is unclear. It appears that central neural inhibition in addition to sex steroid negative feedback contributes to the decrease in fetal gonadotropin concentrations in late gestation. NMDA did not affect fetal oGH or oPRL secretion.     

QSAR modeling and transmission electron microscopy stereology of altered mitochondrial ultrastructure of white blood cells in patients diagnosed as schizophrenic and treated with antipsychotic drugs.

Treatment of patients diagnosed as schizophrenic with antipsychotic drugs (neuroleptics) is known to cause occasional unexplained depletion of white blood cells, especially neutrophil granulocytes. It has been known for many years that neuroleptics can interfere with the mitochondrial respiratory chain in vitro. Because there has been a growing interest recently in mitochondrial targeting of drugs, and since a quantitative structure-activity relationship (QSAR) model that predicts mitochondrial accumulation of neuroleptics has been published, we investigated the effects of neuroleptics on white blood cell mitochondria. Venous blood samples were collected from both patients undergoing treatment with neuroleptics and healthy volunteers. The samples were processed for transmission electron microscopy. The resulting images of white blood cells were analyzed using stereology to compare quantitatively mitochondrial morphology in the patient and control groups. We found that in patients, but not in controls, there was swelling of mitochondria and fragmentation of the mitochondrial cristae. There also were fewer mitochondria in patients than in controls, although due to the swelling of the organelles, the volume density of mitochondria in the two groups was not significantly different. Such changes are typical of a toxic insult. Consequently, it seems plausible that, since schizophrenia is not a disease considered to affect white blood cells per se, these changes probably are due to the medication.     

RAPID SCREENING FOR SALMONELLA IN FISHMEAL BY ENRICHMENT-IMMUNOASSAY

We previously described an enrichment-immunoassay utilizing a T6 monoclonal antibody capture enzyme-linked immunosorbent assay. Here we evaluated it for the rapid screening for Salmonella in fishmeal obtained from the national Animal and Plant Quarantine service in the People's Republic of China. In this method, the number of Salmonella present is first expanded by appropriate enrichment cultures, and the pathogens are then directly detected by the T6 immunoassay. In a total of 94 enrichment cultures of fishmeal, we obtained an overall concordance of 98% between the results obtained in parallel by this method and by conventional test method. The positive prediction by this method was 92% and the negative prediction was 100%. The turn around time for the new test was 27 h which is a significant improvement from the turn around time exceeding 96 h required for the conventional test method. This test proved to be compatible with the routine work flow in the practical setting of a quarantine laboratory.