Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.     

Control of REM sleep: an aspect of the regulation of physiological homeostasis.

Since REM sleep is characterized by a suspension of the hypothalamic integration of homeostatic regulations, it has been assumed that the duration of both REM sleep episodes and of the time interval between the end of one episode and the beginning of the following episode may be regulated according to sleep related processes and the homeostatic needs of the organism. A series of studies performed on the rat has shown that REM sleep episodes occur as two basic types: single REM sleep episodes, that are separated by intervals > 3 min and sequential episodes, that are separated by intervals < or = 3 min and appear in a cluster. Moreover, it has been observed that, in this species, a change in REM sleep occurrence is caused by a modification in the number of episodes and not in their duration. With respect to this, sleep deprivation and recovery are characterized by a decrease and an increase, respectively, in the number of sequential REM sleep episodes, but the number of single episodes tends to be kept constant. The central aspects of this kind of regulation have been examined biochemically in the preoptic-anterior hypothalamus, an area involved in the control of autonomic and sleep related processes. The results show that the accumulation of adenosine 3':5'-cyclic monophosphate (cAMP) is impaired, in this region, during sleep deprivation and appears to return to the control levels, during the recovery, with a rate inversely related to the degree of the previous deprivation. Moreover, it has been observed that the systemic administration of DL-propranolol and LiCl reduces cAMP accumulation mainly in the preoptic-anterior hypothalamus; this condition is concomitant with a reduction in REM sleep occurrence.     

Synthesis, antimicrobial activity and molecular modeling studies of halogenated 4-[1H-imidazol-1-yl(phenyl)methyl]-1,5-diphenyl-1H-pyrazoles

During the course of our studies in the azole antifungals area, we synthesized a number of 1,5-disubstituted 4-[1H-imidazol-1-yl(phenyl)methyl]-1H-pyrazoles, analogues of bifonazole. 1,5-Diphenyl-1H-pyrazole 3 showed weak antimycotic and antibacterial activities in vitro against Candida albicans, Cryptococcus neoformans and Staphylococcus aureus. In order to increase these properties, given that the halo substitution was found to be capable of enhancing antifungal effects, we prepared a series of fluoro and chloro derivatives of 3. The microbiological evaluation carried out on newly synthesized compounds included in vitro assays for antifungal, antibacterial and antimycobacterial activities. Among the tested compounds, some dichloro and trichloro-derivatives showed interesting antimicrobial properties. In particular, compounds 10j,k,l produced inhibitory effects against pathogen representatives of yeast (C. albicans, C. neoformans) and Gram positive bacteria (S. aureus) similar or superior to those of bifonazole. In addition, their activity against Mycobacterium tuberculosis was superior to that of clotrimazole and econazole, which were used as reference drugs. The replacement, in these compounds, of chlorine with fluorine atoms led to inactive derivatives. Docking studies were carried out on the most active compounds, in order to rationalize the pharmacological results.     

In vitro recycling of alpha-D-ribose 1-phosphate for the salvage of purine bases

In this paper, we extend our previous observation on the mobilization of the ribose moiety from a purine nucleoside to a pyrimidine base, with subsequent pyrimidine nucleotides formation (Cappiello et al., Biochim. Biophys. Acta 1425 (1998) 273-281). The data show that, at least in vitro, also the reverse process is possible. In rat brain extracts, the activated ribose, stemming from uridine as ribose 1-phosphate, can be used to salvage adenine and hypoxanthine to their respective nucleotides. Since the salvage of purine bases is a 5-phosphoribosyl 1-pyrophosphate-dependent process, catalyzed by adenine phosphoribosyltransferase and hypoxanthine guanine phosphoribosyltransferase, our results imply that Rib-1P must be transformed into 5-phosphoribosyl 1-pyrophosphate, via the successive action of phosphopentomutase and 5-phosphoribosyl 1-pyrophosphate synthetase; and,in fact, no adenosine could be found as an intermediate when rat brain extracts were incubated with adenine, Rib-1P and ATP, showing that adenine salvage does not imply adenine ribosylation, followed by adenosine phosphorylation. Taken together with our previous results on the Rib-1P-dependent salvage of pyrimidine nucleotides, our results give a clear picture of the in vitro Rib-1P recycling, for both purine and pyrimidine salvage.     

ERK1/2 regulates epidermal chemokine expression and skin inflammation

Resident cell populations of the skin contribute to the inflammatory response by producing an array of chemokines, which attract leukocytes from the circulation. TNF-alpha is a major inducer of proinflammatory mediators in keratinocytes. We have recently observed that epidermal growth factor receptor (EGFR) signaling affects TNF-alpha-driven chemokine expression in epidermal keratinocytes, and its functional impairment increases the levels of crucial chemoattractants such as CCL2/MCP-1, CCL5/RANTES, and CXCL10/IFN-gamma-inducible protein-10. In this study, we report evidence that EGFR-dependent ERK1/2 activity is implicated in this mechanism. Abrogation of ERK1/2 activity with specific inhibitors increased chemokine expression in keratinocytes by enhancing mRNA stabilization. In mouse models, inflammatory response to irritants and T cell-mediated contact hypersensitivity were both aggravated when elicited in a skin area previously treated with an EGFR or a MAPK kinase 1/2 inhibitor. In contrast, impairment of p38alpha beta MAPK phosphorylation markedly attenuated these responses. Our data indicate that EGFR-dependent ERK1/2 activity in keratinocytes takes part to a homeostatic mechanism regulating inflammatory responses, and emphasize the distinct role of MAPKs as potential targets for manipulating inflammation in the skin.     

Characterization of Zebrafish Green Cone Photoresponse Recorded with Pressure-Polished Patch Pipettes,Yielding Efficient Intracellular Dialysis

The phototransduction enzymatic cascade in cones is less understood than in rods, and the zebrafish is an ideal model with which to investigate vertebrate and human vision. Therefore, here, for the first time, the zebrafish green cone photoresponse is characterized also to obtain a firm basis for evaluating how it is modulated by exogenous molecules. To this aim, a powerful method was developed to obtain long-lasting recordings with low access resistance, employing pressure-polished patch pipettes. This method also enabled fast, efficient delivery of molecules via a perfusion system coupled with pulled quartz or plastic perfusion tubes, inserted very close to the enlarged pipette tip. Sub-saturating flashes elicited responses in different cells with similar rising phase kinetics but with very different recovery kinetics, suggesting the existence of physiologically distinct cones having different Ca²⁺ dynamics. Theoretical considerations demonstrate that the different recovery kinetics can be modelled by simulating changes in the Ca²⁺-buffering capacity of the outer segment. Importantly, the Ca²⁺-buffer action preserves the fast response rising phase, when the Ca²⁺-dependent negative feedback is activated by the light-induced decline in intracellular Ca²⁺.     

Reaction to Snakes in Wild Moor Macaques (Macaca maura)

International Journal of Primatology - Snake predation is considered an important evolutionary force for primates. Yet, very few studies have documented encounters between primates and snakes in...