Temperaturadaptation der Sauerstoffbindung des Blutes von Rana esculenta L.

Zusammenfassung Bei einer raschen Temperatursteigerung wird die Leistungsfähigkeit des Blutes hinsichtlich der Sauerstoffaufladung durch eine Rechtsverschiebung der Dissotiationskurve herabgesetzt. Rana esculenta besitzt 2 Regulationen, um diesen Effekt zum Teil zu kompensieren. Überführt man kalt-angepaßte Tiere in mittlere Temperaturen und beläßt sie in diesen, so steigert sich durch einen langsam einsetzenden Anpassungsvorgang der Hämoglobingehalt des Blutes. Nach Umsetzen der Frösche in noch höhere Temperaturen wird durch einen weiteren Regulationsvorgang die Sauerstoffaffinität, d. h. der Sauerstoffgehalt, bezogen auf gleiche Hämoglobinmengen, gesteigert, wodurch die Rechtsverschiebung der Dissoziationskurve teilweise wieder rückgängig gemacht wird. Beide Regulationen sind reversibel.Dissertation bei der Philosophischen Fakultät der Universität Kiel, Teil II (Anregung und Anleitung: Prof. Dr. H. Precht). Die photometrischen Messungen wurden mit einem Pulfrich-Photometer ausgeführt, welches die Deutsche Forschungsgemeinschaft Herrn Prof. Precht zur Verfügung stellte.     

Morphology of the female reproductive organs of the African lion (Panthera leo)

The topography and splanchnology of the reproductive organs of the African lioness were studied and described in situ and after removal. The kidneys were located far caudally in relation to the thirteenth ribs. The suspensory ligament was very well developed, originated in a fan‐like manner from the dorsolateral abdominal wall lateral to the kidney and extended up to a few centimetres cranial to the kidney. The proper ligament of the ovary as well as the round ligament was well developed. The round ligament inserted on the medial femoral fascia. The left ovary was bigger than the right. The ovarian bursa had a short mesosalpinx that did not cover any part of the ovary and the fimbriae extended along the entire length. The urethral tuberculum as well as the urethral crest were well developed. The left uterine horn was longer than the right. The uterine tube was found to open directly into the tip of the uterine horn and not onto a papilla. The reproductive organs of the lioness resembled those of the domestic cat and dog but with some major differences.     

Towards predicting Ca2+‐binding sites with different coordination numbers in proteins with atomic resolution

Ca²⁺‐binding sites in proteins exhibit a wide range of polygonal geometries that directly relate to an equally‐diverse set of biological functions. Although the highly‐conserved EF‐Hand motif has been studied extensively, non‐EF‐Hand sites exhibit much more structural diversity which has inhibited efforts to determine the precise location of Ca²⁺‐binding sites, especially for sites with few coordinating ligands. Previously, we established an algorithm capable of predicting Ca²⁺‐binding sites using graph theory to identify oxygen clusters comprised of four atoms lying on a sphere of specified radius, the center of which was the predicted calcium position. Here we describe a new algorithm, MUG (MUltiple Geometries), which predicts Ca²⁺‐binding sites in proteins with atomic resolution. After first identifying all the possible oxygen clusters by finding maximal cliques, a calcium center (CC) for each cluster, corresponding to the potential Ca²⁺ position, is located to maximally regularize the structure of the (cluster, CC) pair. The structure is then inspected by geometric filters. An unqualified (cluster, CC) pair is further handled by recursively removing oxygen atoms and relocating the CC until its structure is either qualified or contains fewer than four ligand atoms. Ligand coordination is then determined for qualified structures. This algorithm, which predicts both Ca²⁺ positions and ligand groups, has been shown to successfully predict over 90% of the documented Ca²⁺‐binding sites in three datasets of highly‐diversified protein structures with 0.22 to 0.49 Å accuracy. All multiple‐binding sites (i.e. sites with a single ligand atom associated with multiple calcium ions) were predicted, as were half of the low‐coordination sites (i.e. sites with less than four protein ligand atoms) and 14/16 cofactor‐coordinating sites. Additionally, this algorithm has the flexibility to incorporate surface water molecules and protein cofactors to further improve the prediction for low‐coordination and cofactor‐coordinating Ca²⁺‐binding sites. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).     

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca²⁺‐binding pockets of holoproteins to identify such pockets, ignoring possible Ca²⁺‐induced conformational change. In this article, we first report our progress in the analysis of Ca²⁺‐induced conformational changes followed by improved prediction of Ca²⁺‐binding sites in the large group of Ca²⁺‐binding proteins that exhibit only localized conformational changes. The MUG^SR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUG^SR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUG^SR was applied to both X‐ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca²⁺‐binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein.     

Untersuchungen über die Temperaturabhängigkeit von Lebensprozessen bei verschiedenen Wirbellosen

Zusammenfassung Die Atmungsintensität winterruhender Weinbergschnecken (Helix pomatia) weist eine dem Typ 3 entsprechende Temperaturadaptation auf. Der kritische Punkt, von dem ab eine Abhängigkeit des Sauerstoffverbrauches vom Sauerstoffpartialdruck in Erscheinung tritt, wird daher mit steigender Adaptationstemperatur nach niedrigeren Sauerstoffdrucken hin verschoben.Der Atmungsadaptation geht eine entsprechende Aktivitätsanpassung der Dehydrasen in Eiweißdrüse, Mitteldarmdrüse, Fußmuskulatur und Niere parallel. Die Fermentaktivität warmadaptierter Schnecken ist jedoch in den einzelnen Organen in unterschiedlichem Grade gedrosselt. Das Aufwachen aus der Winterruhe ist mit einer Aktivitätssteigerung der Dehydrasen verbunden. Von diesem Prozeß werden die einzelnen Organe zu verschiedenen Zeitpunkten erfaßt, so daß die Aktivität der Succinodehydrase ein und derselben warmadaptierten Schnecke in einigen Organen (Eiweißdrüse und Niere) noch durch die Temperaturadaptation gedrosselt sein kann, während sie in anderen Organen (Fußmuskulatur und Zwitterdrüse) bereits die Aktivität aufgewachter Tiere erreicht.Die Adaptationstemperatur sowie das Aufwachen aus der Winterruhe wirken sich auf den Gehalt an wasserstoffübertragenden Stoffwechselprodukten (Bernsteinsäure) in einigen Organen noch stärker aus als auf die Fermentaktivität. Der Gehalt an gebundenem und freiem Wasser in der Mitteldarmdrüse wird durch die Adaptations temperatur nicht beeinflußt.Auch Lumbriculus variegatus besitzt eine Atmungsadaptation entsprechend Typ 3. Die adaptative Drosselung der Atmungsintensität beginnt hier erst, wenn die Adaptationstemperatur einen bei etwa 14° liegenden Grenzwert überschreitet. Nach Gewöhnung an einen 12stündigen Wechsel von 15 und 23° zeigen die Würmer den gleichen Sauerstoffverbrauch wie nach Anpassung an konstante Temperatur von 19°. Der Umkehrpunkt der Atmungskurve warmadaptierter Tiere liegt bei höherer Temperatur als derjenigen kaltadaptierter. An Gewebesuspensionen konnten weder Atmungsnoch Fermentadaptation (Succinodehydrase) nachgewiesen werden.Sauerstoffverbrauch, Umkehrpunkt der Atmungskurven und Aktivität der Succinodehydrase von Eisenia foetida werden durch die Adaptationstemperatur nicht beeinflußt. Die Aktivität der Katalase sowie die bei den Dehydraseaktivitätsbestimmungen ohne Bernsteinsäurezusatz resultierenden Entfärbungsgeschwindigkeiten nehmen jedoch mit steigender Adaptationstemperatur zu. Die Lebensresistenz gegenüber extrem hohen Temperaturen steigt mit zunehmender Adaptationstemperatur. Umkehrpunkt der Atmungskurve und Hitzetod liegen bei gleicher Temperatur; die gemessene Fermentinaktivierung setzt erst bei höherer Temperatur ein. Die Lebensresistenz gegenüber Hitze ist im Herbst größer als im Frühjahr.Die Stoffwechselintensität von Diapause-Larven von Cephaleia abietis weist ebenfalls keine Temperaturadaptation auf. Die an 24° gewöhnten Larven mit Puppenaugen haben allerdings einen geringeren Sauerstoffverbrauch als die bei kalten und mittleren Temperaturen gehaltenen Tiere. Diese Erscheinung könnte jedoch durch Entwicklungsprozesse bedingt sein. An Gewebesuspensionen aus unterschiedlich adaptierten Diapause-Larven mit Puppenaugen ließen sich keine Unterschiede in der Dehydrasenaktivität nachweisen.Gekürzte Wiedergabe einer Dissertation bei der Philosophischen Fakultät der Universität Kiel, Teil I (Anregung und Anleitung: Prof. Dr. H. Precht).     

Structural differences between Pb2+- and Ca2+-binding sites in proteins: implications with respect to toxicity

Pb²⁺ is known to displace physiologically-relevant metal ions in proteins. To investigate potential relationships between Pb²⁺/protein complexes and toxicity, data from the protein data bank were analyzed to compare structural properties of Pb²⁺- and Ca²⁺-binding sites. Results of this analysis reveal that the majority of Pb²⁺ sites (77.1%) involve 2-5 binding ligands, compared with 6 ± 2 for non-EF-Hand and 7 ± 1 for EF-Hand Ca²⁺-binding sites. The mean net negative charge by site (1.7) fell between values noted for non-EF-Hand (1 ± 1) and EF-Hand (3 ± 1). Oxygen is the dominant ligand for both Pb²⁺ and Ca²⁺, but Pb²⁺ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) Ca²⁺-binding sites. A comparison of binding geometries where Pb²⁺ has replaced Ca²⁺ in calmodulin (CaM) and Zn²⁺ in 5-aminolaevulinic acid dehydratase (ALAD) revealed protein structural changes that appear to be unrelated to ionic displacement. Structural changes observed with CaM may be related to opportunistic binding of Pb²⁺ in regions of high electrostatic charge, whereas ALAD may bind multiple Pb²⁺ ions in the active site. These results suggest that Pb²⁺ adapts to structurally-diverse binding geometries and that opportunistic binding may play an active role in molecular metal toxicity.     

Predicting Ca2+‐binding sites using refined carbon clusters

Identifying Ca²⁺‐binding sites in proteins is the first step toward understanding the molecular basis of diseases related to Ca²⁺‐binding proteins. Currently, these sites are identified in structures either through X‐ray crystallography or NMR analysis. However, Ca²⁺‐binding sites are not always visible in X‐ray structures due to flexibility in the binding region or low occupancy in a Ca²⁺‐binding site. Similarly, both Ca²⁺ and its ligand oxygens are not directly observed in NMR structures. To improve our ability to predict Ca²⁺‐binding sites in both X‐ray and NMR structures, we report a new graph theory algorithm (MUG^C) to predict Ca²⁺‐binding sites. Using carbon atoms covalently bonded to the chelating oxygen atoms, and without explicit reference to side‐chain oxygen ligand co‐ordinates, MUG^C is able to achieve 94% sensitivity with 76% selectivity on a dataset of X‐ray structures composed of 43 Ca²⁺‐binding proteins. Additionally, prediction of Ca²⁺‐binding sites in NMR structures was obtained by MUG^C using a different set of parameters, which were determined by the analysis of both Ca²⁺‐constrained and unconstrained Ca²⁺‐loaded structures derived from NMR data. MUG^C identified 20 of 21 Ca²⁺‐binding sites in NMR structures inferred without the use of Ca²⁺ constraints. MUG^C predictions are also highly selective for Ca²⁺‐binding sites as analyses of binding sites for Mg²⁺, Zn²⁺, and Pb²⁺ were not identified as Ca²⁺‐binding sites. These results indicate that the geometric arrangement of the second‐shell carbon cluster is sufficient not only for accurate identification of Ca²⁺‐binding sites in NMR and X‐ray structures but also for selective differentiation between Ca²⁺ and other relevant divalent cations. © Proteins 2012. © 2012 Wiley Periodicals, Inc.     

Ultrasonographic and laparoscopic evaluation of the reproductive tract of the captive female African lion (Panthera leo)

The use of transabdominal ultrasonography to assess the oestrous cycle has not been previously described in the African lion (Panthera leo). Twelve sexually mature lionesses and five female cubs had their reproductive organs assessed by transabdominal ultrasound. Ovarian findings were compared to laparoscopic findings while performing laparoscopic ovariectomy or salpingectomy. Vaginal cytology was performed and serum progesterone levels were determined. By combining all data the oestrous cycle stage of each lion was determined. One lion was far pregnant and was not operated on. In adults a uterine body could be seen ultrasonographically in 67% of lions while mural structures could be distinguished in 44% of lions. Five uterine horns could be seen in 3 lions. In 12 adults 10 ovaries were found of which eight had discernable follicles or luteal structures. During laparoscopy 12 active ovaries were seen with luteal structures seen in 11 ovaries and follicles in 2 ovaries. Using laparoscopy as the gold standard, ultrasonography had a sensitivity of 66% and specificity of 83% to detect ovarian reproductive activity. Two uterine cysts and a cluster of periovarian cysts were seen in three different lions. Three lions were pregnant, two were in oestrus, three in a luteal phase (dioestrus), and four were in anoestrus. Transabdominal ultrasound in combination with serum progesterone levels and vaginal cytology can be used to assess ovarian cyclical activity with reasonable accuracy in captive bred lions.