Calmodulin-independent coordination of Ras and extracellular signal-regulated kinase activation by Ras-GRF2

Ras-GRF2 (GRF2) is a widely expressed, calcium-activated regulator of the small-type GTPases Ras and Rac. It is a multidomain protein composed of several recognizable sequence motifs in the following order (NH(2) to COOH): pleckstrin homology (PH), coiled-coil, ilimaquinone (IQ), Dbl homology (DH), PH, REM (Ras exchanger motif), PEST/destruction box, Cdc25. The DH and Cdc25 domains possess guanine nucleotide exchange factor (GEF) activity and interact with Rac and Ras, respectively. The REM-Cdc25 region was found to be sufficient for maximal activation of Ras in vitro and in vivo caused Ras and extracellular signal-regulated kinase (ERK) activation independent of calcium signals, suggesting that, at least when expressed ectopically, it contains all of the determinants required to access and activate Ras signaling. Additional mutational analysis of GRF2 indicated that the carboxyl PH domain imparts a modest inhibitory effect on Ras GEF activity and probably normally participates in intermolecular interactions. A variant of GRF2 missing the Cdc25 domain did not activate Ras and functions as an inhibitor of wild-type GRF2, presumably by competing for interactions with molecules other than calmodulin, Ras, and ligands of the PH domain. The binding of calmodulin was found to require several amino-terminal domains of GRF2 in addition to the IQ sequence, and no correlation between calmodulin binding by GRF2 and its ability to directly activate Ras and indirectly stimulate the mitogen-activated protein (MAP) kinase ERK in response to calcium was found. The precise role of the GRF2-calmodulin association, therefore, remains to be determined. A GRF2 mutant missing the IQ sequence was competent for Ras activation but failed to couple this to stimulation of the ERK pathway. This demonstrates that Ras-GTP formation is not sufficient for MAP kinase signaling. We conclude that in addition to directly activating Ras, GRF2, and likely other GEFs, promote the assembly of a protein network able to couple the GTPase with particular effectors.     

Wirkstoffe der B-Gruppe in der Bienennahrung

Summary Through a test (Tribolium-test) which was developed at our institute byK. Offhaus andG. Fröbrich, pollen of ca. 35 and antheres of almost 70 different plants, as well as several honey-proofs and food-sap for the queen could be examined in regard to their effectiveness. The following B-vitamins were tested, in them: B1, B2, B6, -Biotin, Pantothenic Acid, Fol Acid, Nicotin amid, Cholinchlorid, B12 (in honey only!) and the imago-factor.Pollen dispensers, which have been wellknown to the bee-keeper for a long time as a good feeding-ground for the bees (Crocus, Salix, Salvia, Galanthus, etc.), are the best sources for all B-vitamins. First of all among the effectives of the B-group the beekeeper must be interested in the vitamins Pantothenic Acid and -Biotin. They are present in great quantity in the food-sap for the queen and, without doubt, derive from the pollen-food of those bees associated with queen. For the problem of determination, Pantothenic Acid and -Biotin (?) probably play the decisive role. The best sources of Pantothenic Acid for the bees are amongst those plants examined by us: sage, robinia, rose, and lysimachia; and amongst the anemophiles: scleranth-grass and wheat. Then follow the spring-flowers: Crocus, scilla, gagea, narcissus, salix, and among the later flourishing plants: horse-chestnut, iris, nothera, campanula rapuncoloides. Yet 1/4 of all tested plants have pollen with a small percentage of Pantothenic Acid. An especially large quantity of Biotin is found in the antheres of salix and campanula-types. Yet also galanthus, scilla, crocus, gagea, primula, and apple-tree have antheres which contain a great deal of Biotin. In some pollen, respectively antheres, a new factor, necessary for the metamorphosis of Tribolium (and, perhaps, for all other insects too?), was identified, which has also vitamin, character (TIF byFröbrich-Offhaus,Bt byFraenkel).Poisonous for our test subjects were the antheres of philadelphus, digitalis (pollen not poisonous!) and Colchicum. The pollen of the meadow-saffron produces great losses, yet a few insects develop nevertheless, so that it is in question whether this pollen is poisonous.Honey, however, contains only a very small quantity of B-vitamins and those small traces of vitamin probably derive from the pollen which is always mixed with the honey. Folic Acid and Pyridoxin are of the highest percentage, -Biotin and Cholinchlorid follow at a distance—nicotinacid is merely found in traits. Completely negative results were obtained in tests for B₁, B₂, B12 and Pantothenacid.

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Résumé Au moyen d'un test mis au point à notre institut parK. Offhaus etG. Fröbrich, on a pu examiner le spectre d'efficacité du pollen d'environ 35 et des anthères de presque 70 plantes différentes ainsi que celui d'échantillons de miel et de suc nourricier des reines. On y releva les vitamines B suivantes: B₁, B₂, B₆, Biotine , acide pantothénique, acide folique, amide d'acide nicotinique, chloriede-cholin, B₁₂ (seulement dans le miel) et le facteur Imago.Les fournisseurs de pollen, bien connus depuis longtemps de l'apiculteur comme bon terrain de pâture pour les abeilles (Crocus, Salix, Salvia, Galanthus), constituent les sources les meilleures de toutes les vitamines B₁. Parmi les excitants du groupe B, la vitamine acide pantothénique et biotine intéresseront en premier lieu l'apiculteur. On les trouve en quantités particulièrement importantes dans le suc nourricier de la reine, et elles proviennent sans aucun doute de la nourriture faite de pollen des abeilles nourricières. Dans le problème de la détermination, on suppose qu'elles jouent un rôle décisif. Les meilleures sources d'acide pantothénique pour les abeilles parmi les plantes que nous avons examinées à cet égard, sont: salvia, robinier, rose lysimachia, et parmi les anémophiles: scléranthe et froment. Parmi les fleurs de printemps, ce sont:crocus, scilla, gagea, narcissus, salix; parmi les fleurs dont la floraison est plus tardive:aesculus, iris, nothera, campanules rapuncoloides. Le quart des plantes examinées out un pollen avec faible pourcentage d'acide pantothénique. Les anthères desSalix et des différentes variétés de Campanules sont particulièrement riches en biotine. De même, les anthères desgalanthus, scilla, crocus, gagea, primula et pommiers sont relativement riches en biotine. Dans certains pollens, voire même certaines anthères, on a pu prouver l'existence d'un nouveau facteur indispensable pour la métamorphose duTribolium (et peut-être même pour celle de tous les insectes) et présentant par conséquent un caractère vitaminique (TIF chezFröbrich-Offhaus, B_t chezFraenkel).Ont réagi de façon vénéneuse sur la matière du test les anthères duPhiladelphus, digitalis (son pollen est inoffensif), de l'anthirhinum (son pollen est également inoffensif) et ducolchicum. Le pollen du colchique entraîne de nombreux manques; pourtant certains insectes, quoiqu'en petit nombre, se développent, de sorte que la question se pose si ce pollen est vénéneux on non.Par contre, le miel est pauvre en vitamine B et les quelques traces de vitamines que l'on peut relever proviennent vraisemblablement du pollen qui se mêle toujours au miel. L'acide folique et le Pyrodoxin sont encore largement représentés, ensuite viennent la biotine et le chloride cholin; on ne, relève l'amide d'acide nicotinique qu'à l'état de traces. Les expériences recherchant l'existence de B₁, B₂, B₁₂ et d'acide pantothénique se sont montrées totalement négatives.

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Unserm verehrten Lehrer Herrn Prof. Dr. Paul Buchner, zu seinem 70. Geburtstag am 12. April 1956 gewidmet.     

Trois nouveaux alcaloïdes bis-indoliques de Flindersia fo urnieri

Three new bis-indole alkaloids were isolated from Flindersia fournieri. Their structures were elucidated by spectral analysis and chemical correlation with the major alkaloids of the plant.     

Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality

Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform.     

Bacterial Choices for the Consumption of Multiple Resources for Current and Future Needs

Microorganisms differ in their effectiveness in uptake and selection of substances that they bring in from the environment. They also differ in how they balance the allocation of nutrients for immediate and for delayed use. Moreover, they may not take up resources as fast as they seemingly could, and they may extrude derivatives of substances just pumped in. A good deal of these apparent choices must reside in the uptake systems and the linkage of these with the cell’s intermediate metabolism. An important feature is that a resource may vary in concentration from time to time, nutrient to nutrient, and habitat to habitat. This variation must have been critical to the evolution of regulatory processes. Some possibilities for the combined uptake and consumption are considered for substrates serving the same (homologous) and different (heterologous) roles for the bacterium. From the membrane transport processes diagrammed in Fig. 1c and Fig. 2 and corresponding computer program given in Appendix A, the combined effect of uptake processes and cell growth can be studied. The model can be modified for various alternate models to study the possible control of cellular uptake and metabolism for the range of ecological roles of the bacterium.     

Influence of moderate altitude on blood lactate and heart rate in a standardized exercise test in healthy volunteers

Nineteen healthy volunteers were exposed to a standardized exercise test at sea level (SLa), at an altitude of 1700 m before (1700a) and after a moderate 10-day mountain training (1700b), with a final control four weeks later at sea level (SLb). Vital signs, blood lactate and arterial oxygen saturation were determined prior, during or after the exercise test. Whereas systolic blood pressure and heart rate at rest did not change substantially, diastolic blood pressure decreased at the final control (SLb, p<0.05) and oxygen saturation was significantly lower at 1700 m (1700a, 1700b, p<0.01). Lactate at rest increased from 1.16 (SLa) to 1.97 (1700a) mmol/l after acute exposure followed by a slight reduction after adaptation (p<0.05). The mean maximum lactate levels were as follows: 6.03, 10.56, 6.22 and 8.75 (p<0.01). The mean maximum performance increased during the study (225.6, 223.3, 231.6, 248.1 Watt, p<0.01). Lactate versus workload curves did not show a marked shift to the right. No significant changes of maximum heart rates during the exercise test were found. In conclusion, a sojourn at 1700 m provokes an increase of lactate levels with subsequent reduction after acclimatization and has a significant positive impact on the mean maximum performance after moderate mountain training.