Determination of agonist dissociation constants in isolated vasculature: equivalence of estimates obtained by the method of partial irreversible receptor inactivation and a novel application of the operational model of pharmacological agonism

Steady-state contractile responses elicited by activation of the proportional 1-adrenergic and 5-HT2 receptors in isolated rat and rabbit aorta, respectively, were analyzed. Agonist dissociation constants (KA's) obtained by the method of partial irreversible receptor inactivation were compared to KA values determined by fits of the operational model of pharmacological agonism to single concentration response curves (CRCs). The observed nature of the KA estimates obtained with the Furchgott method for phenylephrine (PE) and oxymetazoline (OXY) at the proportional 1-adrenergic receptor and for 5-hydroxytryptamine (5-HT) at the 5-HT2 receptor in isolated rat aorta, and for PE and 5-HT at the proportional 1-adrenergic and 5-HT2 receptors, respectively, in isolated rabbit aorta, was consistent with the hypothesis that the density of membrane receptors is greatly in excess of the density of transducer proteins (i.e., [Ro] much greater than [To]) in these systems. Therefore, KA, efficacy and slope factor estimates were also obtained by computer fits of the operational model to single agonist CRCs in both rat and rabbit aorta, with the empirically determined tissue maximal response (Tmax) substituted for the theoretical parameter Em. In all cases, the mean pKA estimates obtained with the operational model closely approximated and were strongly correlated with the mean pKA estimates determined by the Furchgott method. These studies suggest that, at least in some vascular preparations, Tmax is a good estimate of Em, and moreover, that Em may be not only a specific characteristic of a given receptor-effector system as previously demonstrated by Black and Leff, but that Em may also describe a more general feature of tissue responsiveness that is shared among distinct membrane receptors coupled to similar effector systems. In conclusion, when receptor inactivation studies have indicated that the condition [Ro] much greater than [To] exists, Tmax can be substituted into the operational model to provide valid estimates of agonist KA values at distinct receptor subtypes, in the absence of receptor alkylation.     

Complex polar lipids of a hot spring cyanobacterial mat and its cultivated inhabitants.

The complex polar lipids of the hot spring cyanobacterial mat in the 50 to 55 degrees C region of Octopus Spring, Yellowstone National Park, and of thermophilic bacteria cultivated from this or similar habitats, were compared in an attempt to understand the microbial sources of the major lipid biomarkers in this community. Intact complex lipids were analyzed directly by fast atom bombardment mass spectrometry (FAB-MS), two-dimensional thin-layer chromatography (TLC), and combined TLC-FAB-MS. FAB-MS and TLC gave qualitatively similar results, suggesting that the mat contains major lipids most like those of the cyanobacterial isolate we studied, Synechococcus sp. strain Y-7c-s. These include monoglycosyl, diglycosyl, and sulfoquinosovyl diglycerides (MG, DG, and SQ, respectively) and phosphatidyl glycerol (PG). Though Chloroflexus aurantiacus also contains MG, DG, and PG, the fatty acid chain lengths of mat MGs, DGs, and PGs resemble more those of cyanobacterial than green nonsulfur bacterial lipids. FAB-MS spectra of the lipids of nonphototrophic bacterial isolates were distinctively different from those of the mat and phototrophic isolates. The lipids of these nonphototrophic isolates were not detected in the mat, but most could be detected when added to mat samples. The mat also contains major glycolipids and aminophospholipids of unknown structure and origin. FAB-MS and TLC did not always give quantitatively similar results. In particular, PG and SQ may give disproportionately high FAB-MS responses.     

Vertically stratified frugivore community composition and interaction frequency in a liana fruiting across forest strata

Vertical stratification is a key feature of tropical forests and structures plant–frugivore interactions. However, it is unclear whether vertical differences in plant-frugivore interactions are due to differences among strata in plant community composition or inherent preferences of frugivores for specific strata. To test this, we observed fruit removal of a diverse frugivore community on the liana Marcgravia longifolia in a Peruvian rain forest. Unlike most other plants, Marcgravia longifolia produces fruits across forest strata. This enabled us to study effects of vertical stratification on fruit removal without confounding effects of plant species and stratum. We found a high number of visits of a few frugivore species in the understorey and a low number of visits of many different frugivores in the canopy and midstorey. Whereas partial and opportunistic frugivores foraged across strata with differing frequencies, obligate frugivores were only found eating fruits in the higher strata. Avian frugivores foraging in the canopy were mainly large species with pointed wings, whereas under- and midstorey avian foragers were smaller with rounded wings. Our findings suggest a continuous shift in the frugivore community composition along the vertical gradient, from a few generalized frugivores in the understorey to a diverse set of specialized frugivores in the canopy. This shift in the frugivore community leads to correlated, reciprocal changes from specialized to generalized plant-frugivore interactions. Thus, we conclude that vertical niche differentiation between species in tropical forests persists even when food resources are available across strata. This highlights its role for promoting biodiversity and ecosystem functioning.     

Morphological parameters affecting false lumen thrombosis following type B aortic dissection: a systematic study based on simulations of idealized models

Type B aortic dissection (TBAD) carries a high risk of complications, particularly with a partially thrombosed or patent false lumen (FL). Therefore, uncovering the risk factors leading to FL thrombosis is crucial to identify high-risk patients. Although studies have shown that morphological parameters of the dissected aorta are related to FL thrombosis, often conflicting results have been reported. We show that recent models of thrombus evolution in combination with sensitivity analysis methods can provide valuable insights into how combinations of morphological parameters affect the prospect of FL thrombosis. Based on clinical data, an idealized geometry of a TBAD is generated and parameterized. After implementing the thrombus model in computational fluid dynamics simulations, a global sensitivity analysis for selected morphological parameters is performed. We then introduce dimensionless morphological parameters to scale the results to individual patients. The sensitivity analysis demonstrates that the most sensitive parameters influencing FL thrombosis are the FL diameter and the size and location of intimal tears. A higher risk of partial thrombosis is observed when the FL diameter is larger than the true lumen diameter. Reducing the ratio of the distal to proximal tear size increases the risk of FL patency. In summary, these parameters play a dominant role in classifying morphologies into patent, partially thrombosed, and fully thrombosed FL. In this study, we point out the predictive role of morphological parameters for FL thrombosis in TBAD and show that the results are in good agreement with available clinical studies.

     

Cloning of the amphibolic Calvin cycle/OPPP enzyme d-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) from spinach chloroplasts: functional and evolutionary aspects

Exploiting the differential expression of genes for Calvin cycle enzymes in bundle-sheath and mesophyll cells of the C4 plant Sorghum bicolor L., we isolated via subtractive hybridization a molecular probe for the Calvin cycle enzyme d-ribulose-5-phosphate 3-epimerase (R5P3E) (EC 5.1.3.1), with the help of which several full-size cDNAs were isolated from spinach. Functional identity of the encoded mature subunit was shown by R5P3E activity found in affinity-purified glutatione S-transferase fusions expressed in Escherichia coli and by three-fold increase of R5P3E activity upon induction of E. coli overexpressing the spinach subunit under the control of the bacteriophage T₇ promoter, demonstrating that we have cloned the first functional ribulose-5-phosphate 3-epimerase from any eukaryotic source. The chloroplast enzyme from spinach shares about 50% amino acid identity with its homologues from the Calvin cycle operons of the autotrophic purple bacteria Alcaligenes eutrophus and Rhodospirillum rubrum. A R5P3E-related eubacterial gene family was identified which arose through ancient duplications in prokaryotic chromosomes, three R5P3E-related genes of yet unknown function have persisted to the present within the E. coli genome. A gene phylogeny reveals that spinach R5P3E is more similar to eubacterial homologues than to the yeast sequence, suggesting a eubacterial origin for this plant nuclear gene.Abbreviations R5P3E d-ribulose-5-phosphate 3-epimerase - RPI ribose-5-phosphate isomerase - TKL transketolase - PRK phosphoribulokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - FBP fructose-1,6-bisphophatase - FBP fructose 1,6-bisphosphate - G6PDH glucose-6-phosphate dehydrogenase - 6PGDH 6-phosphogluconate dehydrogenase - OPPP oxidative pentose phosphate pathway - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - FBA fructose-1,6-bisphophate aldolase - IPTG isopropyl -d-thiogalactoside - GST glutathione S-tranferase - PBS phosphate-buffered saline - TPI triosephosphate isomerase     

Tissue specific loss of proliferative capacity of parthenogenetic cells in fetal mouse chimeras

Parthenogenetic cells are lost from fetal chimeras. This may be due to decreased proliferative potential. To address this question, we have made use of combined cell lineage and cell proliferation analysis. Thus, the incorporation of bromodeoxyuridine in S-phase was determined for both parthenogenetic and normal cells in several tissues of fetal day 13 and 17 chimeras. A pronounced reduction of bromodesoxyuridine incorporation by parthenogenetic cells at both developmental stages was only observed in cartilage. In brain, skeletal muscle, heart and intestinal epithelium, this reduction was either less pronounced or observed only at one of the developmental stages analysed. No difference between parthenogenetic and normal cells was observed in epidermis and ganglia. Our results show that a loss of proliferative potential of parthenogenetic cells during fetal development contributes to their rapid elimination in some tissues. The analysis of the fate of parthenogenetic cells in skeletal muscle and cartilage development demonstrated different selection mechanisms in these tissues. In skeletal muscle, parthenogenetic cells were largely excluded from the myogenic lineage proper by early post-midgestation. In primary hyaline cartilage, parthenogenetic cells persisted into adulthood but were lost from cartilages that undergo ossification during late fetal development.     

Distribution of androgenetic cells in fetal mouse chimeras

To asses the potential of androgenetic cells to participate in post-midgestation fetal development we have made use of an in situ detectable cell lineage marker in the analysis of chimeric mouse fetuses containing an androgenetic cell lineage. Our results show conclusively that androgenetic cells participate in the formation of derivatives of all lineages and in some tissues may contribute the majority of the total cell population. However, the allocation or persistence of androgenetic cells was non-random. High contribution of androgenetic cells was observed in brown adipose tissue, mesenchyme, smooth muscle, perichondrium, peripheral nerves and epithelia of the intestinal tract and the trachea. Thus, androgenetic cells were able to efficiently populate mesodermal, ectodermal and endodermal derivatives. In contrast, there was a clear prejudice against androgenetic cells in the brain.     

Alteration of apparent restriction endonuclease recognition specificities by DNA methylases.

An in vitro method of altering the apparent cleavage specificities of restriction endonucleases was developed using DNA modification methylases. This method was used to reduce the number of cleavage sites for 34 restriction endonucleases. In particular, single-site cleavages were achieved for Nhe I in Adeno-2 DNA and for Acc I and Hinc II in pBR322 DNA by specifically methylating all but one recognition sequence.     

Zur Nahrungs?kologie des Wei?storchs (Ciconia ciconia) in Oberschwaben: Beobachtungen an zwei Paaren

Zusammenfassung Das Nahrungssuchverhalten zweier Weißstorch-Paare wurde durch systematische Beobachtung der Störche im Gelände erfaßt. Das Storchenpaar mit gutem Grünlandangebot in der Nähe des Nestes und kleiner Jungenzahl hatte während der ganzen Brutsaison viel Freizeit. Es suchte in einem Entfernungsbereich bis 1,5 km vom Nest Futter und wandte fast ausschließlich die profitable Mäusejagd an. Das Storchenpaar mit schlechtem Grünlandangebot in der Nähe des Nestes und relative großer Jungenzahl nutzte während der Jungenaufzucht einen Großteil der Helligkeitsperiode zur Futtersuche. Es dehnte dabei seinen Entfernungsbereich bis 3,8 km vom Nest aus und ging bei gutem Regenwurmangebot in nahen Entfernungen zum Nest auch der unprofitablen Regenwurmjagd nach. Die Nahrungsaufnahme der Störche betrug während der Brutphase etwa 2600 kJ, während der Aufzucht von ein bis zwei Jungen ungefähr 8850 kJ pro Storch und Tag.

---

On the feeding ecology of the White stork (Ciconia ciconia) in Obserschwaben (Baden-Württemberg, Germany): observations on two pairs

Summary The foraging behaviour of two pairs of White storks was recorded by rigorous observations in the field. One pair of storks, with many meadows in the vicinity of their nest and a small clutch size, spent much time resting throughout the breeding season. They searched for food within a range of 1.5 km from the nest and used the profitable mouse hunting method almost exclusively. When rearing its young, the other pair of storks, with few meadows in the vicinity of their nest and a relatively large clutch size, used a large part of the daylight period for foraging. Thus they expanded their range up to 3.8 km from the nest. When earthworms were abundant, they also used the unprofitable earthworm hunting method within short distances from the nest. The daily energy intake per stork during incubation was approximately 2600 kJ, and approximately 8850 kJ when rearing young.