Structural determinants of salmon calcitonin bioactivity: the role of the Leu-based amphipathic alpha-helix

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu(23),Ala(24)-sCT in which Pro(23) and Arg(24) were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic alpha-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.     

VEGF receptor‐1 involvement in pericyte loss induced by Escherichia coli in an in vitro model of blood brain barrier

The key aspect of neonatal meningitis is related to the ability of pathogens to invade the blood–brain barrier (BBB) and to penetrate the central nervous system. In the present study we show that, in an in vitro model of BBB, on the basis of co‐culturing primary bovine brain endothelial cells (BBEC) and primary bovine retinal pericytes (BRPC), Escherichia coli infection determines changes of transendothelial electrical resistance (TEER) and permeability (Pe) to sodium fluorescein. In the co‐culture model, within BBEC, bacteria are able to stimulate cytosolic and Ca²⁺‐independent phospholipase A₂ (cPLA₂ and iPLA₂) enzyme activities. In supernatants of E. coli‐stimulated co‐cultures, an increase in prostaglandins (PGE₂) and VEGF production in comparison with untreated co‐cultures were found. Incubation with E. coli in presence of AACOCF₃ or BEL caused a decrease of PGE₂ and VEGF release. SEM and TEM images of BBEC and BRPC showed E. coli adhesion to BBEC and BRPC but only in BBEC the invasion occurs. VEGFR‐1 but not VEGFR‐2 blockade by the specific antibody reduced E. coli invasion in BBEC. In our model of BBB infection, a significant loss of BRPC was observed. Following VEGFR‐1, but not VEGFR‐2 blockade, or in presence of AACOCF₃ or BEL, elevated TEER values, reducedpermeability and BRPC loss were found. These data suggest that VEGFR‐1 negatively regulates BRPC survival and its blockade protects the barrier integrity. PGs and VEGF could exert a biological effect on BBB, probably by BRPC coverage ablation, thus increasing BBB permeability. Our results show the role played by the BBEC as well as BRPC during a bacterial attack on BBB. A better understanding of the mechanisms by which E. coli enter the nervous system and how bacteria alter the communication between endothelial cells and pericytes may provide exciting new insight for clinical intervention.     

Ciliate community associated with aquatic macrophyte roots: effects of nutrient enrichment on the community composition and species richness

The objective of this study was to identify the impact of nutrient enrichment on the diversity of the ciliate community associated with the roots of the aquatic macrophyte Eichhornia crassipes. The experiment was performed in the Garças Lake, located in the Upper Paraná River floodplain, Brazil. We conducted two treatments (fertilized and control) with three replicates each. To increase the initial nutrient concentrations in each mesocosm of the fertilized treatment, we added 1000 μg L⁻¹ of KNO₃ and 200 μg L⁻¹ of KH₂PO₄ during each sampling date. We found a relative high number of ciliate species (85 species) and a predominance of hypotrichs. Among the recorded species, about 25% occurred exclusively in the fertilized treatment. Moreover, detrended correspondence analysis demonstrated that the ciliate community associated with E. crassipes roots changed significantly in response to the nutrient input in such a way that the species composition of the fertilized treatment was remarkably different from that of the control. In contrast to our expectations, species richness in the fertilized treatment was significantly higher than that in the control, refuting our hypothesis that species richness decreases under eutrophic conditions.     

Molecular modeling study for the binding of zonisamide and topiramate to the human mitochondrial carbonic anhydrase isoform VA

Zonisamide and topiramate are two antiepileptic drugs known to induce weight loss in epilepsy patients. These molecules were recently shown to act as carbonic anhydrase (CA) inhibitors, being presumed that the weight loss may be due to the inhibition of the mitochondrial isozymes CA VA and CA VB involved in metabolic processes, among which lipid biosynthesis. To better understand the interaction of these compounds with CAs, here, we report a homology modeling and molecular dynamics simulations study on their adducts with human carbonic anhydrase VA (hCA VA). According to our results, in both cases the inhibitor sulfamate/sulfonamide moiety participates in the canonical interactions with the catalytic zinc ion, whereas the organic scaffold establishes a large number of van der Waals and polar interactions with the active site cleft. A structural comparison of these complexes with the corresponding homologues with human carbonic anhydrase II (hCA II) provides a rationale to the different affinities measured for these drugs toward hCA VA and hCA II. In particular, our data suggest that a narrower active site cleft, together with a different hydrogen bond network arrangement of hCA VA compared to hCA II, may account for the different Kd values of zonisamide and topiramate toward these physiologically relevant hCA isoforms. These results provide useful insights for future design of more isozyme-selective hCA inhibitors with potential use as anti-obesity drugs possessing a novel mechanism of action.     

Structure of the Bateman2 domain of yeast Snf4: dimeric association and relevance for AMP binding

AMP-activated protein kinase (AMPK) is a central regulator of energy homeostasis in mammals. AMP is believed to control the activity of AMPK by binding to the gamma subunit of this heterotrimeric enzyme. This subunit contains two Bateman domains, each of which is composed of a tandem pair of cystathionine beta-synthase (CBS) motifs. No structural information is currently available on this subunit, and the molecular basis for its interactions with AMP is not well understood. We report here the crystal structure at 1.9 Angstrom resolution of the Bateman2 domain of Snf4, the gamma subunit of the yeast ortholog of AMPK. The structure revealed a dimer of the Bateman2 domain, and this dimerization is supported by our light-scattering, mutagenesis, and biochemical studies. There is a prominent pocket at the center of this dimer, and most of the disease-causing mutations are located in or near this pocket.     

Vanadate inhibits blue light-stimulated swelling of vicia guard cell protoplasts

When supplied under low chloride concentrations, vanadate inhibits the blue light-stimulated swelling of Vicia faba L. guard cell protoplasts in a dose-dependent fashion. The volume of guard cell protoplasts incubated in 10 mm K-imino-diacetic acid, 0.4 m mannitol, and 1 mm CaCl₂ remained essentially constant under 1000 μmol m⁻² s⁻¹ red light, but increased an average of 27% after 8 min of the addition of 50 μmol m⁻² s⁻¹ blue light to the background red light. At 500 μm, vanadate completely inhibits the response to blue light. Vanadate also inhibits the swelling of guard cell protoplasts stimulated by the H⁺-ATPase agonist fusicoccin. The vanadate sensitivity of the blue light-stimulated swelling implicates a proton-pumping ATPase as a component of the sensory transduction of blue light in guard cells.     

Solution structure of nociceptin peptides.

Peptides embedded in the sequence of pre-pro-nociceptin, i.e. nociceptin, nocistatin and orphanin FQ2, have shed light on the complexity of the mechanisms involving the peptide hormones related to pain and have opened up new perspectives for the clinical treatment of pain. The design of new ligands with high selectivity and bioavailability, in particular for ORL1, is important both for the elucidation and control of the physiological role of the receptor and for their therapeutic importance. The failure to obtain agonists and antagonists when using, for nociceptin, the same substitutions that are successful for opioids, and the conformational flexibility of them all, justify systematic efforts to study the solution conformation under conditions as close as possible to their natural environment. Structural studies of linear peptides in solution are hampered by their high flexibility. A direct structural study of the complex between a peptide and its receptor would overcome this difficulty, but such a study is not easy since opioid receptors are membrane proteins. Thus, conformational studies of lead peptides in solution are still important for drug design. This review deals with conformational studies of natural pre-nociceptin peptides in several solvents that mimic in part the different environments in which the peptides exert their action. None of the structural investigations yielded a completely reliable bioactive conformation, but the global conformation of the peptides in biomimetic environments can shed light on their interaction with receptors.     

A fruit-specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria x ananassa) fruit ripening

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry ( Fragaria × ananassa ) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.     

Tonoplast vesicles of Beta vulgaris storage root show functional aquaporins regulated by protons

BACKGROUND INFORMATION: Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re-evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation. RESULTS: An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped-flow technique. The preparation showed high water permeability (485 microm x s(-1)), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl(2) (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western-blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition. CONCLUSIONS: Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl(2), not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell-to-cell pathway.     

The water permeability of Arabidopsis plasma membrane is regulated by divalent cations and pH

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (P(f)) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher P(f) and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their P(f ) and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of P(f) at 50-100 microM free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of P(f )at pH 7.2-7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.