Metabolic rate of Arctic charr eggs depends on their parentage

The metabolic rate (specific heat output) of individual eyed-stage eggs of Arctic charr Salvelinus alpinus (Linnaeus, 1758) originating from different families was measured with direct microcalorimetry. Metabolic rates varied between 2.3–7.9 μW ind⁻¹ and 0.06–0.22 μW mg⁻¹. Absolute heat output was unrelated to egg size, but size-scaled or specific heat output was negatively correlated with egg size, measured as diameter, dry mass or fresh mass. Metabolic rates varied significantly between families, suggesting that genetic and/or maternal effects affect embryonic metabolism in Arctic charr. Heat output increased almost linearly from 3.4 to 16.7 μW ind⁻¹ (0.09–0.67 μW mg⁻¹) during the embryonic development. Although the metabolic rate varied between the families and egg metabolic rate increased during development, there was an unexpected disconnect between metabolic rate and hatching time.     

Quantitative variations of individual carotenoids in relationship with the leaflet development of six species of the genus Ceratozamia (Cycads)

The content and relative variations of individual carotenoids during the leaflet development stages (I, II, III, A and P) of six species of Ceratozamia (Cycads) were investigated. There is an unusual, transitory and marked presence of six red stroma keto-carotenoids in the first development stages, while the thylakoidal carotenoids showed a low concentration during the same period. As no official A1cm1% coefficients were available, it was necessary to calculate these for the following stroma carotenoids: semi-beta-carotenone (major component), triphasiaxanthin, ceratoxanthin, ceratozamiaxanthin, kuesteriaxanthin and ceratoxanthone. The stroma keto-carotenoids, which reached the highest content in the first development stage (average of 78% of total carotenoids), were always present in the five species: C. fuscoviridis, C. robusta, C. spinosa, C. kuesteriana and C. hildae, but never in C. mexicana. From stage II, the stroma keto-carotenoids decreased and finally disappeared in the green adult leaflets. The thylakoidal carotenoids showed a minimum at stage III, and then increased to a maximum in the perennial leaflets. Among these, beta-carotene showed an anomalous and characteristic behaviour, being a minor component during leaflet development (from stage I to A). In stage P it was markedly exceeded not only by lutein but also by alpha-carotene, neoxanthin and violaxanthin, and in C. robusta also by lutein-5,6-epoxide. Additionally, the alpha/beta ratio in these species is unusual: it increased from 0.3-0.5 to 1.5-3.0 during leaflet development. Moreover, antheraxanthin amounts are very small, while zeaxanthin was present only in the evergreen leaflets of C. mexicana in small quantities. Lutein-5,6-epoxide represented more than 5% of thylakoidal carotenoids in the leaflets of all the species. A revision of the taxonomic rank of C. fuscoviridis is recommended.     

Structure of doubly prenylated Ypt1:GDI complex and the mechanism of GDI-mediated Rab recycling

In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

Microassays for a set of enzymes in individual small marine copepods

Fluorogenic assays for a set of five hydrolytic enzymes involved in digestion and food utilization (alanine and arginine aminopeptidase, lipase/esterase, chitobiase, and beta-glucosidase) were optimized to measure activities of these enzymes in the same extracts of individual small North Sea copepods. The enzyme activities of Acartia clausi, Centropages typicus, Corycaeus anglicus, Paracalanus parvus, and Temora longicornis showed distinct species specific activity patterns, but also high intra-specific variability. Protein, lipids, carbon and nitrogen (C, N) were determined with micro-scale assays in individual copepods or in batches of 10 to 50 animals. Water soluble protein contents ranged from 16% to 38%, and lipid contents from 2.4% to 5.5% of dry mass. The molar C/N ratios were between 4.1 and 4.5. The presented microassays provide suitable tools for studying physiological reactions of copepods and other small pelagic crustaceans in response to variable environmental conditions.     

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.     

Subcellular localization and regulation of type-1C and type-5 phosphodiesterases

We investigated the subcellular localization of PDE5 in in vitro human myometrial cells. We demonstrated for the first time that PDE5 is localized in discrete cytoplasmic foci and vesicular compartments corresponding to centrosomes. We also found that PDE5 intracellular localization is not cell- or species-specific, as it is conserved in different animal and human cells. PDE5 protein levels are strongly regulated by the mitotic activity of the smooth muscle cells (SMCs), as they were increased in quiescent, contractile myometrial cultures, and conditions in which proliferation was inhibited. In contrast, PDE1C levels decreased in all conditions that inhibited proliferation. This mirrored the enzymatic activity of both PDE5 and PDE1C. Increasing cGMP intracellular levels by dbcGMP or sildenafil treatments did not block proliferation, while dbcAMP inhibited myometrial cell proliferation. Together, these results suggest that PDE5 regulation of cGMP intracellular levels is not involved in the control of SMC cycle progression, but may represent one of the markers of the contractile phenotype.     

Photo-ionization spectroscopy and mass spectrometry of some molecular and supramolecular asymmetric systems in the isolated state

Asymmetric molecular and supramolecular systems are characterized by: i. the circular dicroism in the angular distribution of valence photoelectrons emitted from randomly oriented chiral molecules by their interaction with circularly polarized VUV light; ii. the different stability and reactivity of diastereomeric aggregates. Both these aspects may have some relationship with the "chiral enrichment mechanism" of chirogenesis, based on the preferential destruction of one enantiomer of a racemate by interaction with a chiral agent, whether a massive species or a circularly polarized photon. The most recent spectroscopic and mass spectrometric studies on this topic are reported in the present mini-review.     

Bleaching and stress in coral reef ecosystems: hsp70 expression by the giant barrel sponge Xestospongia muta

Sponges are a prominent component of coral reef ecosystems. Like reef-building corals, some sponges have been reported to bleach and die. The giant barrel sponge Xestospongia muta is one of the largest and most important components of Caribbean coral reef communities. Tissues of X. muta contain cyanobacterial symbionts of the Synechococcus group. Two types of bleaching have been described: cyclic bleaching, from which sponges recover, and fatal bleaching, which usually results in sponge death. We quantified hsp70 gene expression as an indicator of stress in X. muta undergoing cyclic and fatal bleaching and in response to thermal and salinity variability in both field and laboratory settings. Chlorophyll a content of sponge tissue was estimated to determine whether hsp70 expression was related to cyanobacterial abundance. We found that fatally bleached sponge tissue presented significantly higher hsp70 gene expression, but cyclically bleached tissue did not, yet both cyclic and fatally bleached tissues had lower chlorophyll a concentrations than nonbleached tissue. These results corroborate field observations suggesting that cyclic bleaching is a temporary, nonstressful state, while fatal bleaching causes significant levels of stress, leading to mortality. Our results support the hypothesis that Synechococcus symbionts are commensals that provide no clear advantage to their sponge host. In laboratory experiments, sponge pieces incubated at 30 °C exhibited significantly higher hsp70 expression than control pieces after 1.5 h, with sponge mortality after less than 15 h. In contrast, sponges at different salinities were not significantly stressed after the same period of time. Stress associated with increasing seawater temperatures may result in declining sponge populations in coral reef ecosystems.