Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, exerts a variety of effects on the gastrointestinal tract. The present study was aimed at evaluating the possible implication of N/OFQ in the maintenance of gastric mucosal integrity. N/OFQ was given either centrally or peripherally 30 min prior to intragastric administration (i.g.) of 1 ml/rat of ethanol (either 25% or 50%, v/v), which produces macroscopically visible gastric lesions. Intracerebroventricular (i.c.v.) injection of 2 microg/rat of N/OFQ significantly reduced lesions caused by 50% ethanol, while 1 microg/rat was enough to significantly reduce lesions caused by 25% ethanol. Intracerebroventricular injection of 5 microg/rat of the selective NOP receptor antagonist, UFP-101, completely reversed the protective effect of N/OFQ, 1 or 4 microg/rat against 25% or 50% ethanol, respectively. The intraperitoneal (i.p.) injection of N/OFQ produced a significant reduction of lesions induced by 50% ethanol, the peak effect being observed at 10 microg/kg. Intraperitoneal pretreatment with UFP-101, 120 microg/kg, completely abolished the protective effect of peripherally injected N/OFQ. Therefore, N/OFQ acts both centrally and peripherally as a protective agent against ethanol-induced gastric lesions, and its effect is mediated by NOP receptors.     

Carbonic anhydrase inhibitors. Zonisamide is an effective inhibitor of the cytosolic isozyme II and mitochondrial isozyme V: solution and X-ray crystallographic studies

The antiepileptic drug zonisamide was considered to act as a weak inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) (with a K(I) of 4.3 microM against the cytosolic isozyme II). Here we prove that this is not true. Indeed, testing zonisamide in the classical assay conditions of the CO2 hydrase activity of hCA II, with incubation times of enzyme and inhibitor solution of 15 min, a K(I) of 10.3 microM has been obtained. However, when the incubation between enzyme and inhibitor was prolonged to 1 h, the obtained K(I) was of 35.2 nM, of the same order of magnitude as that of the clinically used sulfonamides/sulfamates acetazolamide, methazolamide, ethoxzolamide and topiramate (K(I)s in the range of 5.4-15.4 nM). The inhibition of the human mitochondrial isozyme hCA V with these compounds has been also tested by means of a dansylamide competition binding assay, which showed zonisamide and topiramate to be effective inhibitors, with K(I)s in the range of 20.6-25.4 nM. The X-ray crystal structure of the adduct of hCA II with zonisamide has also been solved at a resolution of 1.70 A, showing that the sulfonamide moiety participates in the classical interactions with the Zn(II) ion and the residues Thr199 and Glu106, whereas the benzisoxazole ring is oriented toward the hydrophobic half of the active site, establishing a large number of strong van der Waals interactions (<4.5 A) with residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202.     

Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.     

Novel anellated pyrazoloquinolin-3-ones: synthesis and in vitro BZR activity

A series of pyrazolo[4,3-c]pyrrolo[3,2-f]quinolin-3-one derivatives 6, 7a-c, 8a,b, 9a,b and 10-12 were synthesized as modified pyrazoloquinolinone analogs (PQs) and evaluated for their ability to inhibit radioligand to central and peripheral benzodiazepine receptors (BZRs) and their effect on GABA(A) alpha1beta2gamma2L receptors expressed in Xenopus laevis oocytes. Multistep synthesis starting from 5-nitroindole, via the Gould-Jacobs reaction to the quinoline nucleus, yielded key intermediates 9-chloro-3H-pyrrolo[3,2-f]quinoline-8-carboxylates. The reaction of the latter with methyl-hydrazine and various phenyl-hydrazines furnished the final compounds. In order to confirm the expected tetracyclic 2-substituted-2H-pyrazolopyrroloquinolin-3-one structure, IR spectrophotometric, mono-1H and 13C and bi-dimensional spectrometric and HRMS analyses were carried out: all compounds were found to be 2-substituted 3-keto tautomers; compound 6 only differed because it turned out to be 1-methyl-2H-pyrazolo[4,3-c]pyrrolo[3,2-f]quinolin-3-olo. The results of this work are consistent with those previously reported for PQs: 7-9 show high potency in displacing specific [3H]flunitrazepam from its receptor site; no compound was active in inhibiting the binding of [3H]PK 11195. They all act as antagonists at central BZR.     

Innate immunity and inflammation in ageing: a key for understanding age-related diseases

The process of maintaining life for the individual is a constant struggle to preserve his/her integrity. This can come at a price when immunity is involved, namely systemic inflammation. Inflammation is not per se a negative phenomenon: it is the response of the immune system to the invasion of viruses or bacteria and other pathogens. During evolution the human organism was set to live 40 or 50 years; today, however, the immune system must remain active for much a longer time. This very long activity leads to a chronic inflammation that slowly but inexorably damages one or several organs: this is a typical phenomenon linked to ageing and it is considered the major risk factor for age-related chronic diseases. Alzheimer's disease, atherosclerosis, diabetes and even sarcopenia and cancer, just to mention a few – have an important inflammatory component, though disease progression seems also dependent on the genetic background of individuals. Emerging evidence suggests that pro-inflammatory genotypes are related to unsuccessful ageing, and, reciprocally, controlling inflammatory status may allow a better chance of successful ageing. In other words, age-related diseases are "the price we pay" for a life-long active immune system: this system has also the potential to harm us later, as its fine tuning becomes compromised. Our immune system has evolved to control pathogens, so pro-inflammatory responses are likely to be evolutionarily programmed to resist fatal infections with pathogens aggressively. Thus, inflammatory genotypes are an important and necessary part of the normal host responses to pathogens in early life, but the overproduction of inflammatory molecules might also cause immune-related inflammatory diseases and eventually death later. Therefore, low responder genotypes involved in regulation of innate defence mechanisms, might better control inflammatory responses and age-related disease development, resulting in an increased chance of long life survival in a "permissive" environment with reduced pathogen load, medical care and increased quality of life.     

The preferred conformation of the tripeptide Ala-Phe-Ala in water is an inverse gamma-turn: implications for protein folding and drug design

Recent studies have provided evidence that peptides as short as tripeptides do adopt preferred conformations. Here we report that the tripeptide Ala-Phe-Ala (AFA) in aqueous solution preferentially forms an inverse gamma-turn. Circular dichroism (CD) indicated the presence of a predominant turn structure, and Fourier transform infrared (FTIR) bands suggested the presence of a gamma-turn forming a bifurcated H-bond with the solvent molecules. The high-resolution structure was obtained by a combined use of NMR spectroscopy and calculations. On the basis of 30 unambiguous ROESY-derived distance restraints (including the Halpha-NH NOE between Ala(1) and Ala(3) and a hydrogen bond between the CO group of Ala(1) and the NH group of Ala(3)), calculations clearly demonstrated the presence of an inverse gamma-turn centered on Phe(2). From NOE data, we estimated a mole fraction for the gamma-turn of 0.65. Since for AFA an extended beta-strand was also reported [Eker, F., Griebenow, K., Cao, X., Nafie, L. A., and Schweitzer-Stenner, R. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 10054-10059], we investigated the possibility that gamma-turn and beta-strand may represent two major conformations. By using a best-fit procedure that calculated experimental NOEs as weighted averages of the effects originating from both structures, we were able to calculate with good accuracy the backbone NOEs at 280 K in terms of the two limiting conformers, yielding a mole fraction for the gamma-turn and beta-strand conformations of 0.60 and 0.40, respectively, in good agreement with those found by NOE data. The implication of the existence of a preferred conformation by a small structural element is discussed in the context of the nucleation of protein folding events and the design of small peptide and peptidomimetic drugs.     

A substrate-induced switch in the reaction mechanism of a thermophilic esterase: kinetic evidences and structural basis

The reaction mechanism of the esterase 2 (EST2) from Alicyclobacillus acidocaldarius was studied at the kinetic and structural level to shed light on the mechanism of activity and substrate specificity increase previously observed in its double mutant M211S/R215L. In particular, the values of kinetic constants (k1, k(-1), k2, and k3) along with activation energies (E1, E(-1), E2, and E3) were measured for wild type and mutant enzyme. The previously suggested substrate-induced switch in the reaction mechanism from kcat=k3 with a short acyl chain substrate (p-nitrophenyl hexanoate) to kcat=k2 with a long acyl chain substrate (p-nitrophenyl dodecanoate) was validated. The inhibition afforded by an irreversible inhibitor (1-hexadecanesulfonyl chloride), structurally related to p-nitrophenyl dodecanoate, was studied by kinetic analysis. Moreover the three-dimensional structure of the double mutant bound to this inhibitor was determined, providing essential information on the enzyme mechanism. In fact, structural analysis explained the observed substrate-induced switch because of an inversion in the binding mode of the long acyl chain derivatives with respect to the acyl- and alcohol-binding sites.     

Human DNA polymerases lambda and beta show different efficiencies of translesion DNA synthesis past abasic sites and alternative mechanisms for frameshift generation

Human DNA polymerases (pols) beta and lambda could promote template slippage and generate -1 frameshifts on defined heteropolymeric DNA substrates containing a single abasic site. Kinetic data demonstrated that pol lambda was more efficient than pol beta in catalyzing translesion DNA synthesis past an abasic site, particularly in the presence of low nucleotide concentrations. Moreover, pol lambda was found to generate frameshifts in two ways: first, by using a nucleotide-stabilized primer misalignment mechanism, or second, by promoting primer reannealing using microhomology regions between the terminal primer sequence and the template strand. Our results suggest a molecular mechanism for the observed high in vivo rate of frameshifts generation by pol lambda and highlight the remarkable ability of pol lambda to promote microhomology pairing between two DNA strands, further supporting its proposed role in the nonhomologous end joining process.