3,154 research outputs found
Chloride interaction in the SLC6 amino acid cotransporter KAAT1
No full textThe SLC6 family of solute transporters groups eukaryotic, Cl--dependent proteins as the serotonin transporter SERT (1) and the GABA transporter GAT1 (2) and prokaryotic, Cl--independent proteins, as the family model LeuT (3, 4). The main role of the anion in the transport cycle appears to be related to the neutralization of the positive charges of sodium ions transported with the substrate. KAAT1 is a lepidopteran SLC6 amino acid cotransporter, activated by Na+ and K+, but characterized by a weak chloride dependence (5, 6). In recent years, taking advantage of the special features of KAAT1, we have investigated the structural/functional relationships within the SLC6 family. By site-directed mutagenesis and functional expression in Xenopus laevis oocytes, we have identified residues involved in Na+ and K+ interaction and in amino acid translocation (7,8,9). The aim of this study has been the identification of the molecular determinants of chloride interaction of KAAT1 to obtain insights in the transport mechanism of SLC6 members. Comparison of KAAT1 sequence with SERT, GAT1 and LeuT has revealed some differences in residues forming the putative anion binding site but, among theme, only T339 seemed to be relevant for chloride dependence: T339S and T339E mutant transport activity became indeed almost completely Cl--dependent. The mutation of a further residue (T67), conserved only in KAAT1 and in the other weakly Cl--dependent transporter of the family CAATCH1, affected KAAT1 activity: T67Y mutant was fully chloride independent whereas T67S and T67A showed an enhancement in chloride dependence. In order to confirm the role of T67, we built the reciprocal mutant of KAAT1 T67Y in GAT1 transporter: Y60T, interestingly, showed a reduced chloride dependence compared to the wt. These data suggest that T67 and T339 influence KAAT1 interaction with chloride.
1. Forrest L.R. et al., Proc Natl Acad Sci U S A. 2007; 104:12761-6.
2. Zomot E. et al., Nature. 2007 Oct 11; 449: 726-30.
3. Yamashita A. et al., Nature. 2005; 437: 215-23
4. Ben-Yona A. et al., J Biol Chem. 2011; 286: 2826-33
5. Castagna M. et al., Proc Natl Acad Sci U S A.; 95: 5395-400
6. Bettè S. et al., Channels (Austin). 2008; 2: 358-62
7. Mari et al., Cell. Mol. Life Sci. 2004, 61: 243-56.
8. Miszner et al., J. Physiol. 2007, 58: 1899-1913.
9. Castagna et al., Am. J. Physiol. Cell. Physiol. 2007, 293: C1286-C129
A donor-acceptor Stenhouse adduct active in neuronal GABAARs and photoswitchable in water
No full text(PO 8) Donor-acceptor Stenhouse adducts are a rapidly emerging class of photochromic compounds whose conformation can be efficiently switched using visible and nearinfrared light. Over the last year, the switching mechanism of DASA has been extensively studied. However, the use of these compounds in water has been hampered by a spontaneous and irreversible conversion to a non photoactive form. The difficulty of using DASAs in water is even more disappointing since they are native red absorbing photoswitching, quality that is widely appreciated in the field of photopharmacology. This field combines a pharmacological approach with the use of light to enable spatio-temporal control of biological processes and drug action. To date, photopharmacology has been extensively used to manipulate biological activity at the cellular level by targeting ion channels, G protein-coupled receptors, enzymes and protein–protein interactions [1,2]. In order to reduce phototoxicity of light used in photopharmacology, low-energy light (i.e. red light) should be used (less scattering in tissue and deeper penetration in the body) and red-absorbing photodrugs need to be designed. To this end, here, we present the molecular design of a novel γ-aminobutyric acid type A receptors (GABAARs) ligand derivative based on the DASA scaffold that displays photochromic properties with red light and is active in neuronal GABAARs [3].
References
1. Eli, S.; Castagna, R.; Mapelli, M.; Parisini, E. Front. Mol. Biosci. 2022, 9, 841777.
2. Castagna, R.; Kolarski, D.; Cuttoli, R. D.-d.; Maleeva, G. J. Mol. Neurosci. 2022, 72, 1433.
3. Castagna, R.; Maleeva, G.; Pirovano, D.; Matera, C.; Gorostiza, P. chemRxiv 2022. doi:10.26434/ chemrxiv-2022-0ps7
The SLC6/NSS family members KAAT1 and CAATCH1 are weakly Cl--dependent
No full textKAAT1 and CAATCH1, amino acid transporters cloned from the intestine of the lepidoptera Manduca sexta (Castagna et al., 1998, PNAS, 95: 5395-5400; Feldman et al., 2000, J. Biol Chem., 275: 24518-24526), are members of the SLC6/NSS family, which groups neurotransmitter, aminergic transmitter, amino acid and osmolyte transporters coupled to Na+, K+ and Cl- gradients.
KAAT1 and CAATCH1 are able to transport different amino acids depending on the contransported cation (Na+ or K+) but their Cl--dependence is not completely defined yet.
The Cl- binding site of two members of the family, the serotonin transporter SERT (Forrest et al., 2007, PNAS, 104: 12761-12766) and the GABA transporter GAT1 (Zomot et al., 2007, Nature, 449: 726-730), has been recently modelled on the basis of their functional properties and by structural homology to the leucine transporter LeuT, a prokaryotic member of the family whose three-dimensional structure has been solved at atomic level (Yamashita et al., 2005, Nature, 437: 215-223). The analyses have highlighted the role of a serine residue, that in the Cl--independent LeuT corresponds to Glu 290, and of an asparagine (Asn 286) that also contributes to the coordination of Na+ in the Na1 binding site of LeuT.
With the aim to clarify the role exerted by chloride in SLC6/NSS transporters, the Cl--dependence of KAAT1 and CAATCH1 have been here investigated by the expression in Xenopus laevis oocytes and the measurement of induced amino acid uptakes.
Despite KAAT1 and CAATCH1 posses the same residue of serine (Ser342, KAAT1 numbering) present in strictly chloride dependent transporters, their transport activities resulted weakly Cl--dependent compared to GAT1. By analysis of the structure of Cl--dependent (SERT and GAT1) and Cl--independent (LeuT) transporters, we selected several residues present in the putative Cl- binding site of KAAT1 and investigated their involvement in chloride interaction
A theory of contracts for web services
No full text<p>Contracts are behavioural descriptions of Web services. We devise a theory of contracts that formalises the compatibility of a client to a service, and the safe replacement of a service with another service. The use of contracts statically ensures the successful completion of every possible interaction between compatible clients and services.</p>
<p>The technical device that underlies the theory is the definition of filters, which are explicit coercions that prevent some possible behaviours of services and, in doing so, they make services compatible with different usage scenarios. We show that filters can be seen as proofs of a sound and complete subcontracting deduction system which simultaneously refines and extends Hennessy's classical axiomatisation of the must testing preorder. The relation is decidable and the decision algorithm is obtained via a cut-elimination process that proves the coherence of subcontracting as a logical system.</p>
<p>Despite the richness of the technical development, the resulting approach is based on simple ideas and basic intuitions. Remarkably, its application is mostly independent of the language used to program the services or the clients. We also outline the possible practical impact of such a work and the perspectives of future research it opens.</p>
Sex differences in aerobic fitness in top-class soccer referees
No full textCastagna, C, Bizzini, M, D'Ottavio, S, and Araújo Póvoas, SC. Sex differences in aerobic fitness in top-class soccer referees. J Strength Cond Res 32(11): 3216-3221, 2018-The aim of this study was to assess the aerobic-fitness differences between male and female top-class soccer field referees (FRs). This with the purpose to provide cutoff values useful for training prescription in female FRs. Forty female top-class FRs (age 34.18 ± 3.50 years and 5 ± 3.9 years international refereeing experience) and 52 male FRs (age 38.4 ± 3.3 years and 5 ± 3.5 years international refereeing experience) candidates in the preliminary open list developed by the FIFA Refereeing Department for the 2014 and 2015 World Cup Tournaments, participated in the study. The FRs were tested for aerobic fitness under laboratory conditions with a progressive speed treadmill test until exhaustion. Female FRs showed to possess, on average, lower (large effect) levels of aerobic fitness and performance compared with their male counterparts. The female FRs' V[Combining Dot Above]O2max (48.1 ± 4.4 ml·kg·min) was 7% (large effect) lower than the male FRs (51.9 ml·kg·min). Peak treadmill speed was 11% lower (large effect) in female FRs (16.27 ± 0.94 vs. 14.64 ± 0.96 km·h). The receiver operating characteristic curve analysis enabled cutoff values (47.8 ml·kg·min for V[Combining Dot Above]O2max) that may be used as preliminary cues to guide physiological selection and training prescription in female FRs aiming to officiate male-soccer matches. Only 2.5% of the female FRs showed V[Combining Dot Above]O2max higher than the mean values of male FRs when using the scaled notation (0.68). Female FRs aiming to officiate male competitions should consider training intensities at anaerobic threshold speed (13 km·h, 95% heart rate max) when developing aerobic fitness. Given the very large sex differences in aerobic performance, strength/power training should be proposed to perspective female top-class FRs
In onda. Saggi su donne e radiodrammi in Inghilterra dagli anni Ottanta ad oggi
No full textIn onda includes four essays on radio plays written by English female authors Marina Warner, Eva Figes, Selma Dabbagh and Timberlake Wertenbaker. The book analyses both original works for the radio and adaptations of classics and modern classics. The author examines the affirmation of women's voices and family relationships and conflicts
Functional role of a highly conserved sequence motif in the insect amino acid transporter KAAT1
No full textKAAT1 is an insect amino acid transporter, member of the NSS/SLC6 family of solute carriers, with peculiar functional properties, being activated by Na+ and by K+ (Castagna et al., PNAS, 1998). KAAT1 sequence shows a stretch of three consecutive glycines (Gly85 - Gly87) that is highly conserved between members of the family and that, according to topology prediction algorithms and to the crystal structure of the NSS/SLC6 member LeuT (Yamashita et al., Nature, 2005), is located in the extracellular loop 1 (EL1), near the access of permeation pathway.
In the present study we have investigated the functional role of this region by KAAT1 site directed mutagenesis and expression in X. laevis oocytes. The substitution of each glycine with alanine determined a reduction of transport activity of 40 - 50 % compared with wild-type (wt), both in the presence of a Na+ or a K+ gradient. The shift of the glycine stretch toward the N or the C terminus of the protein, obtained by the synthesis of the double mutants N84G/G87A and G85A/A88G, reduced the activity respectively to 13 and 8 % of wt, whereas the single mutants N84G and A88G showed a residual transport activity that was respectively 40 % and 5 % of wt.
Our results indicate that EL1 residues influence KAAT1 activity and that in particular, the conserved three-glycine stretch constitutes a “functional motif” in which not the single glycine residue, but the position of the entire motif plays an essential role in transport function
Underwriting Fees and Power Derivatives
No full textIn this paper we analyze the problem of determining standby underwriting fees within the framework of option-pricing theory. Financial institutions that provide standby underwriting for a stock placement bear the risk of having to buy unplaced stocks if the offered quantity is not completely absorbed by the market. We describe a simple model in which the equilibrium price of the stock at the end of the placement period is log-normal and the demand curve for stocks can shift according to stochastic shocks. We show that the value of the guarantee offered by the financial institutions is proportional to the value of a quadratic power put, that is a derivative whose payoff is the square of an ordinary put. This option can be priced within the Black & Scholes theoretical framework. The closed-form formula shows that the value of a quadratic put is much greater than that of an ordinary put and is not simply the square of the latters value. The paper also analyzes the problem of reinsurance, which arises when the underwriting fee is partially transferred to another financial
institution that provides a subsidiary guarantee. We analyze three different contractual arrangements
between two financial institutions, and accordingly show how to allocate the total fee consistently
between them. A simulated application of the model is also presented
Game activity and blood lactate in men's elite water-polo players
No full textThe purpose of this study was to examine game demands during highly competitive men’s water-polo games after the introduction of the new Federation International Natation Association (FINA)
rules. Participants of this study were 77 outfield international
level men’s water-polo players (age 25.9 +/- 4.5 years, height
188 +/- 4.5 cm, body mass 93 +/- 10.9 kg). Game earlobe blood
lactate, speed, and distance covered (semiautomatic image
recognition system) were measured during 6 highly competitive
matches (FINA International Tournament). In the game, players
covered 1,613 +/- 150 m (n = 68). This corresponds to a mean
coverage rate of 54 6 5.8 mmin21 (n = 68). At speeds faster
than 1.4 m/s (high-intensity swimming), players covered 44%
of the total distance. A significant decrease in coverage rate
was detected during the final stage of the game. Mean blood lactate concentration was 7.7 +/- 1.0 mmol/l (range 2.2–14.3). Center Defenders (1,816 +/- 496 m) covered significantly more distance swimming compared to Field (1,676 +/- 348 m) and Center-Forward (1,317 +/- 281 m) players. Mean match blood lactate concentrations for Center Forwards, Center Defenders, and Field Players were 11.2 +/- 1.0 (3–14.3), 6.7 +/- 0.9 (1.4–8), and 5.3 +/- 0.9 mmol/l (2.2–11.9), respectively. This study confirmed the high-intensity nature of male elite level water polo showing remarkable roledependent game demands. As a result, strength and conditioning interventions should be individualized and mainly address intermittent high-intensity endurance and anaerobic fitness
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