Characterization of Mycobacterium tuberculosis NAD kinase: functional analysis of the full-length enzyme by site-directed mutagenesis

NAD kinase is the only known enzyme catalyzing the formation of NADP, a coenzyme implicated in most reductive biosynthetic reactions and in many antioxidant defense systems. Despite its importance, nothing is known regarding its structure or mechanism of catalysis. Mycobacterium tuberculosis NAD kinase has been overexpressed in Escherichia coli and purified to homogeneity. The molecular and kinetic properties of the enzyme resulted in significant differences from those reported by others on a proteolytically degraded form of the protein. Indeed the full-length enzyme displays an allosteric behavior and shows a strict preference for inorganic polyphosphate as the phosphate donor. It is inhibited by the reaction product NADP and by both NADH and NADPH. The mycobacterial enzyme shares with all other known NAD kinases a highly conserved region (spanning residues 189-210), particularly rich in glycines, which differs from the primary sequences of all previously identified nucleotide-binding sites. Alanine-scanning mutagenesis performed on 11 conserved residues within this domain revealed its importance in catalysis. A total of 6 of 11 mutated proteins completely lost the enzymatic activity while retaining the same oligomeric state of the wild-type protein, as demonstrated by gel-filtration analysis. Substitutions of S199 and G208 with alanine rendered enzyme versions with reduced activity. Their kinetic characterization, performed on purified proteins, revealed kinetic parameters toward ATP and polyphosphate similar to those of the wild-type enzyme. On the contrary, when the kinetic analysis was performed by using NAD as the variable substrate, significant differences were observed with respect to both the allosteric behavior and the catalytic efficiency, suggesting that the mutated region is likely involved in NAD binding.     

20S proteasome mediated degradation of DHFR: implications in neurodegenerative disorders

The 20S proteasome is responsible for the degradation of protein substrates implicated in the onset and progression of neurodegenerative disorders, such as alpha-synuclein and tau protein. Here we show that the 20S proteasome isolated from bovine brain directly hydrolyzes, in vitro, the dihydrofolate reductase (DHFR), demonstrated to be involved in the pathogenesis of neurodegenerative diseases. Furthermore, the DHFR susceptibility to proteolysis is enhanced by oxidative conditions induced by peroxynitrite, mimicking the oxidative environment typical of these disorders. The results obtained suggest that the folate metabolism may be impaired by an increased degradation of DHFR, mediated by the 20S proteasome.     

Appetite regulation: The central role of melatonin in Danio rerio

Melatonin is the hormonal mediator of photoperiodic information to the central nervous system in vertebrates and allows the regulation of energy homeostasis through the establishment of a proper balance between energy intake and energy expenditure. The aim of this study was to evaluate the role of melatonin in appetite central control analyzing the involvement of this hormone in the regulation of feeding behavior in the zebrafish Danio rerio. For this purpose, the effect of two different melatonin doses (100 nM and 1 μM) administered for 10 days, via water, to zebrafish adults was evaluated at both physiological and molecular level and the effect of melatonin was considered in relation to the most prominent systems involved in appetite regulation. For the first time, in fact, melatonin control of food intake by the modulation of leptin, MC4R, ghrelin, NPY and CB1 gene expression was evaluated.The results obtained indicate that melatonin significantly reduces food intake and the reduction is in agreement with the changes observed at molecular level. A significant increase in genes codifying for molecules involved in feeding inhibition, such as leptin and MC4R, and a significant reduction in the major orexigenic signals including ghrelin, NPY and CB1 are showed here.Taken together these results support the idea that melatonin falls fully into the complex network of signals that regulate food intake thus playing a key role in central appetite regulation.     

c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-beta1 and 4-hydroxynonenal in colon mucosa

Cells of colonic mucosa are sensitive to the Smad-mediated growth-inhibitory effect of transforming growth factor-beta1 (TGF-beta1). Another important cell growth inhibitor is the polyunsaturated lipid peroxidation end product, 4-hydroxynonenal (HNE), which triggers apoptosis through c-Jun N-terminal kinase (JNK) activation. Interestingly, a close association between TGF-beta1 and HNE was found in the progression of human colon cancer, with concentration of both molecules inversely related to the malignancy. We investigated the cross talk between Smads and JNK signal transduction pathways in inducing apoptosis. To this purpose TGF-beta1 and HNE were added singly or in combination to CaCo-2 human colon adenocarcinoma cells. The cotreatment induced a marked enhancement of apoptosis and of JNK and Smad4 activities much more than either individual molecule. Cell preincubation with the JNK inhibitor SP600125 significantly prevented JNK and Smad4 enhancement and, subsequently, the cooperative proapoptotic effect was abolished. The primary role of JNK activity in TGF-beta1/HNE cooperative signaling was fully confirmed in a second set of experiments by using JNKi I, a more selective kinase inhibitor. Hence, in tumor cells becoming resistant to TGF-beta1-mediated growth inhibition, increased induction of the remaining TGF-beta1 pathways by interaction with other antiproliferative molecules, such as HNE, could help in inhibiting tumor growth.     

The enantiomers of epiboxidine and of two related analogs: synthesis and estimation of their binding affinity at α4β2 and α7 neuronal nicotinic acetylcholine receptors

Epiboxidine hydrochlorides (+)-2 and (-)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(-)-3 and (+)-4/(-)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes.     

Differences in kainate receptor involvement in hippocampal mossy fibre long-term potentiation depending on slice orientation

Long-term potentiation (LTP) is a well-established experimental model used to investigate the synaptic basis of learning and memory. LTP at mossy fibre - CA3 synapses in the hippocampus is unusual because it is normally N-methyl-d-aspartate (NMDA) receptor-independent. Instead it seems that the trigger for mossy fibre LTP involves kainate receptors (KARs). Although it is generally accepted that pre-synaptic KARs play an essential role in frequency facilitation and LTP, their subunit composition remains a matter of significant controversy. We have reported previously that both frequency facilitation and LTP can be blocked by selective antagonism of GluK1 (formerly GluR5/Glu(K5))-containing KARs, but other groups have failed to reproduce this effect. Moreover, data from receptor knockout and mRNA expression studies argue against a major role of GluK1, supporting a more central role for GluK2 (formerly GluR6/Glu(K6)). A potential reason underlying the controversy in the pharmacological experiments may reside in differences in the preparations used. Here we show differences in pharmacological sensitivity of synaptic plasticity at mossy fibre - CA3 synapses depend critically on slice orientation. In transverse slices, LTP of fEPSPs was invariably resistant to GluK1-selective antagonists whereas in parasagittal slices LTP was consistently blocked by GluK1-selective antagonists. In addition, there were pronounced differences in the magnitude of frequency facilitation and the sensitivity to the mGlu2/3 receptor agonist DCG-IV. Using anterograde labelling of granule cells we show that slices of both orientations possess intact mossy fibres and both large and small presynaptic boutons. Transverse slices have denser fibre tracts but a smaller proportion of giant mossy fibre boutons. These results further demonstrate a considerable heterogeneity in the functional properties of the mossy fibre projection.     

Synthesis and binding affinity at α4β2 and α7 nicotinic acetylcholine receptors of new analogs of epibatidine and epiboxidine containing the 7-azabicyclo[2.2.1]hept-2-ene ring system

A group of novel racemic nicotinic ligands structurally related to epibatidine or epiboxidine [(±)-10-(±)-17] was synthesized through a palladium-catalyzed cross-coupling between the appropriate vinyl triflate and a range of organometallic heterocycles. The target compounds were evaluated for binding affinity at the α4β2 and α7 neuronal nicotinic receptors (nAChRs). The set of 3-pyridinyl derivatives (±)-10, (±)-11 and (±)-12 exhibited an affinity for the α4β2 nAChR subtype in the subnanomolar range (K(i) values of 0.20, 0.40 and 0.50nM, respectively) and behaved as α4β2 versus α7 subtype selective ligands. Interestingly, the epiboxidine-related dimethylammonium iodide (±)-17, which retained a good affinity for the α4β2 nAChR (K(i)=13.30nM), tightly bound also to the α7 subtype (K(i)=1.60nM), thus displaying a reversal of the affinity trend among the reference and new nicotinic ligands under investigation.     

Arundo micrantha,a new reed species for Italy,threatened in the freshwater habitat by the congeneric invasive A. donax

Arundo micrantha Lam., a reed species from southern Mediterranean area, has found in Sardinia. Its presence represents the first record for Italy and the third one for western Europe. Investigations on distribution and ecology of the Sardinian population have been carried out, with special focusing on synecology of the species. A. micrantha has found in 40 localities mainly distributed along temporary streams and permanent rivers in the central and southern part of the island. Most of Sardinian populations occurs on alluvial soils in Thermomediterranean bioclimatic context. The autonomy of Sardinian phytocoenoses was characterized with the definition of exclusive floristic taxa, biological and chorological elements, and syndinamic relationships with other assemblages. Comparing the community with other reed beds from the Mediterranean basin, a marked floristic differentiation arose between the phytocoenoses characterized by autochthonous Arundo species and those dominated by the invasive Arundo donax. Finally, management activities are proposed to ensure the conservation of this threatened autochthonous plant community in freshwater habitat.