Fecal cortisol radioimmunoassay to monitor adrenal gland activity in the bottlenose dolphin (Tursiops truncatus) under human care

Fecal glucocorticoid measurement is an important noninvasive tool to monitor animal health. A radioimmunoassay (RIA) method was developed to measure fecal cortisol in bottlenose dolphins under human care. The method was used to measure baseline hormone levels and evaluate the adrenal response to environmental challenges in a small number of individual dolphins. The method was validated by precision and accuracy tests and by comparison with liquid chromatography‐mass spectrometry (LC‐MS). The parallelism test suggested few matrix interferences. The assay showed a good degree of precision within assay (CV = 5.4%) and between assays (CV = 4.1%). The RIA significantly correlated with the LC‐MS method (r = 0.838, P < 0.01). The recovery test and the comparison between RIA and LC‐MS suggested that the RIA slightly underestimates fecal cortisol concentrations, although the degree of accuracy was good. This study established that bottlenose dolphins excrete appreciable amounts of fecal cortisol (healthy subjects: 0.2–9.5 ng/g). Therefore, chronic HPA axis activation may be monitored in fecal samples by immunoassays after validating a suitable extraction protocol. The RIA could discriminate conditions of stimulation (pregnancy, parturition, isolation, transportation) and inhibition (diazepam administration) of the HPA axis and may, therefore, be useful for monitoring dolphin well‐being.     

Stress protein of cyanobacteria CP36: interaction with photoactive complexes and formation of supramolecular structures

Cyanobacterium Anacystis nidulans R2, Synechocystis sp. PCC 6803 (wild-type strain and mutants Delta2 and Delta3 lacking PSII and PSI, respectively), and Synechocystis sp. BO 9201 synthesize the pigment--protein complex CP36 (CPIV-4, CP43') under iron deficiency in the medium. Accumulation of CP36 is accompanied by structural reorganizations in the photosynthetic membranes. Integrating mean times of excitation relaxation (quenching) are 2.2 nsec (CP36), 1 nsec (PSI), and 420 psec (PSII in Fm state). The energy migration between CP36 and the photosystems can be described by a model of a one-layer ring of CP36 around core-complexes. The excitation from CP36 to PSI is transferred within <10 psec. The energy transfer from CP36 to PSII occurs during 170 psec. Cells with low content of CP36 probably contain only a latent fraction of unbound to phycobilisomes PSII which is the analog of PSIIbeta of higher plants. In PSI there are four binding sites for CP36 monomers per RC. PSII can bind up to 32 molecules of CP36 per RC. Cells with a large amount of CP36 contain monomer form of PSII core-complex which can bind eight tetramers of CP36 (8 binding sites). In conditions of iron deficiency only one monomer of a dimer PSII core-complex is destroyed and released chlorophyll is accumulated in CP36. Accumulation of CP36 in A. nidulans cells can be accompanied by membrane stacking which is similar to the stacking in chlorophyll b-containing organisms. The stacking can occur in the region of localization of PSII latent fraction bound to CP36. The membrane stacking shields PSII stromal surfaces from the aqueous phase for activation of electron transfer on the acceptor side of PSII.     

Glucose decreases respiratory control ratio in EL-4 tumor cells.

EL-4 ascites thymoma cells are shown to have high aerobic glycolysis and decreased Pasteur effect. At the same time, glucose produces a much smaller inhibitory effect on cell respiration (Crabtree effect) than in Ehrlich ascites carcinoma (EAC) cells. In intact EL-4 cells, the respiratory control ratio (RCR) was found to be 6.2 with endogenous substrates and 8.0 with glutamine. Glucose decreased the RCR to 3.2, by stimulating the state 4 respiration. In rat thymocytes and EAC cells, such an effect of glucose was absent (RCR of 7.0 and 7.2, respectively). It is suggested that in EL-4 tumor cells, the high aerobic glycolysis and small Crabtree effect may be due to glucose-induced 'uncoupling' of oxidation and phosphorylation.     

Carotenoids of Eriobotrya japonica

The carotenoids of the loquat fruit Eriobotrya japonica Golden Nugget variety, were investigated. They were identified according to their chromatographic, spectrophotometric and chemical properties and compared with standard pigments. For some of the carotenoids, MS were determined. Pulp and peels were investigated separately. The main pattern of the pulp carotenoids was β-carotene (33%), γ-carotene (6%), cryptoxanthin (22%), lutein, violaxanthin and neoxanthin, each about 3–4%. The peel, with a carotenoid content 5 times as high, had a similar pattern, but the ratio between the main pigments differed: β-carotene (50%); γ-carotene (5%); cryptoxanthin (5%); lutein (13%); violaxanthin, neoxanthin, 3–4%. The carotenoids of the loquat (subfamily Maloideae) were very similar to those of the apricot (Prunus armeniaca-subfamily Prunoideae) both of the family of Rosaceae. The intergeneric differences are more pronounced, which is of possible taxonomic significance. The lower concentration of cryptoxanthin and the high concentration of lutein in the peels is noteworthy and of biosynthetic interest.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Distinct hsp70 domains mediate apoptosis-inducing factor release and nuclear accumulation

Although hsp70 antagonizes apoptosis-inducing factor (AIF)-mediated cell death, the relative importance of preventing its release from mitochondria versus sequestering leaked AIF in the cytosol remains controversial. To dissect these two protective mechanisms, hsp70 deletion mutants lacking either the chaperone function (hsp70-deltaEEVD) or ATPase function (hsp70-deltaATPase) were selectively overexpressed before exposing cells to a metabolic inhibitor, an insult sufficient to cause mitochondrial AIF release, nuclear AIF accumulation, and apoptosis. Compared with empty vector, overexpression of wild type human hsp70 inhibited bax activation and reduced mitochondrial AIF release after injury. In contrast, mutants lacking either the chaperone function (hsp70-deltaEEVD) or the ATP hydrolytic domain (hsp70-deltaATPase) failed to prevent mitochondrial AIF release. Although hsp70-deltaEEVD did not inhibit bax activation or mitochondrial membrane injury after cell stress, this hsp70 mutant co-immunoprecipitated with leaked AIF in injured cells and decreased nuclear AIF accumulation. In contrast, hsp70-deltaATPase did not interact with AIF either in intact cells or in a cell-free system and furthermore, failed to prevent nuclear AIF accumulation. These results demonstrate that mitochondrial protection against bax-mediated injury requires both intact chaperone and ATPase functions, whereas the ATPase domain is critical for sequestering AIF in the cytosol.     

Triggering aggresome formation. Dissecting aggresome-targeting and aggregation signals in synphilin 1

Abnormal polypeptides that escape proteasome-dependent degradation and aggregate in cytosol can be transported via microtubules to an aggresome, a recently discovered organelle where aggregated proteins are stored or degraded by autophagy. We used synphilin 1, a protein implicated in Parkinson disease, as a model to study mechanisms of aggresome formation. When expressed in na?ve HEK293 cells, synphilin 1 forms multiple small highly mobile aggregates. However, proteasome or Hsp90 inhibition rapidly triggered their translocation into the aggresome, and surprisingly, this response was independent on the expression level of synphilin 1. Therefore, aggresome formation, but not aggregation of synphilin 1, represents a special cellular response to a failure of the proteasome/chaperone machinery. Importantly, translocation to aggresomes required a special aggresome-targeting signal within the sequence of synphilin 1, an ankyrin-like repeat domain. On the other hand, formation of multiple small aggregates required an entirely different segment within synphilin 1, indicating that aggregation and aggresome formation determinants can be separated genetically. Furthermore, substitution of the ankyrin-like repeat in synphilin 1 with an aggresome-targeting signal from huntingtin was sufficient for aggresome formation upon inhibition of the proteasome. Analogously, attachment of the ankyrin-like repeat to a huntingtin fragment lacking its aggresome-targeting signal promoted its transport to aggresomes. These findings indicate the existence of transferable signals that target aggregation-prone polypeptides to aggresomes.     

ATPase activity of the heat shock protein hsp72 is dispensable for its effects on dephosphorylation of stress kinase JNK and on heat-induced apoptosis

A major inducible heat shock protein, Hsp72, has previously been found to stimulate dephosphorylation (inactivation) of stress kinase JNK in heat-shocked cells and protect them from apoptosis. Using Rat-1 fibroblasts with constitutive expression of a human Hsp72 or its deletion mutant lacking an ATPase domain (C-terminal fragment (CTF)), we tested whether ATPase activity of Hsp72 is necessary for these effects. We found that expression of CTF markedly increased, similarly to the intact protein, JNK dephosphorylation in heat-shocked cells. As a result, JNK inactivation following heat shock occurred much faster in cells expressing either full-length or mutant Hsp72 than in parental cells and this was accompanied by suppression of heat-induced apoptosis. Thus, protein refolding activity of Hsp72 appears to be dispensable for its effect on JNK inactivation and apoptosis.     

Ultrasonographic appearance of tissue is a better indicator of CL function than CL diameter measurement in dairy cows

In this study, the ovaries of 99 randomly selected Friesian cows were examined by ultrasonography measuring the diameter and evaluating the appearance of corpora lutea (CLs) in order to assess the most reliable method for their functional classification. Concurrently, blood samples were taken and analyzed for plasma progesterone (P4) concentration. On the basis of the ultrasonographic measurement of the diameter of the CL, three groups were established: (A) CL not detected (n = 30), (B) CL psi < 20 mm (n = 22), and (C) CL psi > or = 20mm (n = 47). On the basis of the ultrasonographic appearance, three different groups were established: (A) CL not detected (n = 30), (B) evolving CL (n = 25), and (C) mid-cycle CL (n = 44). On the basis of the P4 values, CLs were functionally classified in the following three groups: (A) CL not detected when plasma P4 was lower than 1 ng/ml (n = 27), (B) evolving CL when plasma P4 was between 1 and 4 ng/ml inclusive (n = 29), and (C) mid-cycle CL when plasma P4 was more than 4 ng/ml (n = 43). The degree of agreement between plasma P4 concentrations and either ultrasonographic classification (diameter or appearance) was highly significant (P < 0.001). However, the results of the present study suggest that for the evaluation of functional classification of the CL in cows ultrasonographic appearance is more reliable than the evaluation of the diameter.