Untersuchungen über die lichtabhängige Carotinoidsynthese

Summary The purpose of these studies was to find which steps in the biosynthetic pathway of carotenoids in Fusarium are under photoregulation. After separation by column chromatography -carotene, neurosporene, -carotene, torulene, neurosporaxanthin and lycopene were identified from their absorption spectra in visible light and by co-chromatography tests with carotenoids from other organisms. No other carotenoids were detected. These components were each present in trace amounts (0.5–2 g/g dry weight) in stricly dark grown cultures. During incubation of the mycelium in buffered glucose solutions in darkness these carotenoids accumulated slowly but linearly with time.After a lag period of 30 min following photoinduction a sequential increase of the carotenoids occurs in the order mentioned above. With the exception of lycopene this sequence follows a decreasing degree of saturation. Addition of cycloheximide at various intervals during the incubation after illumination results in a differential inhibition of the synthesis of the various carotenoids, the pattern of which closely resembles the time sequence of increase of the carotenoids following photoinduction. It is therefore concluded that the synthesis of all these carotenogenic enzymes is photoinduced.Incubation of the mycelium with diphenylamine inhibits carotenoid synthesis; however, after illumination lower concentrations cause an increase in the amount of the more unsaturated components. Incubation with H₂O₂ results in the same time sequence of increase of the carotenoids as after photoinduction, whereas in incubations with mercuribenzoate the pattern is different. Synthesis of sterols (probably ergosterol) is not increased after illumination but is slightly decreased. From these results we conclude that the first step of the biosynthetic pathway on which photoregulation takes place lies between farnesyl-pyrophosphate and the coloured carotenoids.

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Die Untersuchungen wurden in großzügiger Weise von der Deutschen Forschungsgemeinschaft unterstützt.     

Untersuchungen zur Synchronisierbarkeit einzelner Pigmentmangel-Mutanten von Chlorella

Zusammenfassung Verschiedene Chlorella-Mutanten, die Chlorophyll a und b (Mutanten 10 und 11), je nach Kulturbedingungen nur Chlorophyll a oder Chlorophyll a und b (Mutante 41) oder nur Spuren von Chlorophyllen (Mutante 31) enthielten, wurden auf ihr Verhalten unter synchronisierenden Kulturbedingungen untersucht.Eine optimale Synchronisation war in einem Licht-Dunkel-Wechsel von 10 Licht- zu 14-Dunkelstunden zu erzielen.Die Synchronisationsschärfe war relativ gering. Unter keiner der angewendeten Kulturbedingungen ließ sich eine Vollsynchronisation erzielen; der Synchronisationstyp war am besten mit einer Gruppen-synchronisation zu vergleichen, bei der laufend Zellen von der einen in die andere Gruppe überwechseln.Unabhängig vom Vorhandensein von Chlorophyll a oder b und vom Ausmaß organischer Zusätze zu den Nährlösungen zeigten alle untersuchten Chlorella-Mutanten den gleichen Synchronisationstyp.Da die untersuchten Mutanten trotz verschiedener Pigmentzusammensetzung bis hin zum praktischen Chlorophyllverlust in gleicher Weise synchronisierbar sind, ist eine Beteiligung der Chlorophylle am Zeitgeber-Mechanismus als sehr unwahrscheinlich anzusehen.

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Studies on synchronization of some pigment-deficient Chlorella mutants

Summary Four different mutant strains of Chlorella pyrenoidosa were studied under conditions giving rise to synchronized mass cultures. The mutants contained either both chlorophyll a and b, only chlorophyll a or only traces of green pigments.Optimal synchronization was found to occur under a light-dark-regiment of 10:14 hours. It was impossible to achieve a complete synchronization; most of the cells developed autospores only every second cycle. This behavior was independent from the pigmentation of the strain; this was taken as evidence for the assumption, that the chlorophylls are not engaged in the timing process.

     

Flexibility of Acanthamoeba myosin rod minifilaments.

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.     

Non-invasive detection of the single motor unit action potential by averaging the spatial potential distribution triggered on a spatially filtered motor unit action potential.

For research as well as diagnostic applications the non-invasive detection of the activity of single motor units is of interest. The most direct information is expected to be found in monopolarly recorded data. But when an array of surface electrodes is used for the monopolar recordings of the potential distribution on the skin, in most cases an additional invasive needle electrode is utilized to detect the exact points in time when a certain motor unit is firing. With this supplementary information, an averaging of the monopolar EMG tracings can be performed. In this paper, a completely non-invasive methodology is presented which replaces the invasive needle by a spatial filtering procedure. The EMG signals from the m. biceps brachii are recorded monopolarly with an electrode array. Afterwards, a spatial filtering procedure, called normal double differentiating filter, is applied to the data. The EMG signals obtained are investigated by means of an amplitude threshold to distinguish the activity of different motor units. The point of the maximum amplitude of the selected peaks then is used as trigger point to average the monopolar EMG data. The time courses of the motor unit action potential signals found after applying the described procedure show similar shapes, while two different components are to be identified: corresponding to the spread of the excitation, one is referring to stationary, the other to travelling events. These results justify the possibility to replace the needle electrode to obtain a trigger event in the future by the non-invasive spatial filtering procedure.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Differences in hydration coupled to specific and nonspecific competitive binding and to specific DNA Binding of the restriction endonuclease BamHI

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120-150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.