NAD-dependent inhibition of the NAD-glycohydrolase activity in A549 cells

NAD glycohydrolases are enzymes that catalyze the hydrolisis of NAD to produce ADP-ribose and nicotinamide. Regulation of these enzymes has not been fully elucidated. We have identified an NAD-glycohydrolase activity associated with the outer surface of the plasma membrane in human lung epithelial cell line A549. This activity is negatively regulated by its substrate -NAD but not by -NAD. Partial restoration of NADase activity after incubation of the cells with arginine or histidine, known ADP-ribose acceptors, suggests that inhibition be regulated by ADP-ribosylation. A549 do not undergo to apoptosis upon NAD treatment indicating that this effect be likely mediated by a cellular component(s) lacking in epithelial cells.     

Massive apoptosis of colonocytes induced by butyrate deprivation overloads resident macrophages and promotes the recruitment of circulating monocytes

Our previous investigations demonstrated a rapid, massive apoptosis of colonocytes after butyrate deprivation. However, while in vitro apoptotic bodies and cells were sludged at the epithelial surface, in vivo they were phagocytosed by the resident macrophages. In the present study the guinea pig colon was perfused in vivo in the presence or absence of butyrate with the aim of identifying the cells involved in the removal of apoptotic material and the method of clearance. Morphological, immunohistochemical and DNA fragmentation analyses were applied. The results demonstrated massive apoptosis of colonocytes in the absence of butyrate. The resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections they phagocytosed and transported apoptotic material from the epithelial intercellular spaces into their bodies. Apparently, the macrophages could not cope with the great amount of apoptotic material they had to eliminate: the recruitment of circulating monocytes occurred. This was revealed by the application of antibodies directed against MAC 387, CD68 (PG-M1), and S-100, which detected distinct monocyte/macrophage populations in the lamina propria. The recruited cells were phenotypically different from resident macrophages, their occurrence being typical in inflamed tissues. In conclusion, butyrate deprivation in vivo led to untimely death of colonocytes and triggered changes in the lamina propria indicative of an inflammatory response.     

An investigation of the seasonal pattern of mannitol content in deciduous and evergreen species of the oleaceae growing in northern Sicily

In several species of the Oleaceae, mannitol, already present at considerable levels, accumulates in response to stress. This family comprises both deciduous and evergreen species, and we investigated the role of mannitol in deciduous malacophyll and evergreen sclerophyll species growing under the same conditions in the field. The relationship between mannitol content and changes in rainfall or temperature was also studied. The mannitol content of leaves of Fraxinus ornus L., F. angustifolia Vahl., Olea europaea L. and Phillyrea media L. was determined by gas chromatography. Leaf samples were collected once a month for 1 year. In the two ash species, the seasonal pattern of mannitol content appeared the same: a gradual increase in spring, peaking in summer, followed by a gradual decrease. The mannitol content was similar in both species, ranging between 260 and 720 micromol g(-1) d. wt. The seasonal pattern of mannitol content in Olea and Phillyrea was similar for both species, but unlike that of Fraxinus did not show a summer peak. Rainfall was negatively correlated with the seasonal increase of mannitol content in ash. Mannitol content increased gradually during drought, reaching a maximum value at the end of the dry season. Temperature did not have a direct influence on mannitol content. In Olea and Phillyrea, variations in mannitol content were poorly correlated with rainfall or temperature, indicating that mannitol does not have a primary role in the response of these species to the hot, dry summer conditions.     

Unfolding of pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase probed by time-resolved tryptophan fluorescence

Proteins utilizing pyridoxal 5'-phosphate as a coenzyme constitute a large superfamily and are currently classified into three functional groups and five structural fold types. Despite the variability of sequences and catalyzed reactions, they share relevant structural, dynamic and functional properties. Therefore, they constitute an optimal system to investigate the relative influence of primary sequence and coenzyme interactions on folding pathways, structural stability and enzymatic function. O-Acetylserine sulfhydrylase is a dimeric pyridoxal 5'-phosphate dependent enzyme that catalyzes the synthesis of L-cysteine from O-acetylserine and sulfide. The time-resolved fluorescence study of O-acetylserine sulfhydrylase unfolding, here reported, indicates that the coenzyme stabilizes the protein structure. The dependence on denaturant concentration of tryptophan lifetimes in the holo- and apo-enzyme demonstrates that the interactions with the coenzyme stabilize the C-terminal domain to a higher extent with respect to the N-terminal domain. This result is discussed in terms of a linkage between the differential stabilization brought about by the coenzyme and the different degrees of conformational flexibility required by the specialized functional role of distinct protein regions.     

Ribosomal localization of translation initiation factor IF2

Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit's factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.     

Donor-site morbidity after free vascularized autogenous fibular transfer: subjective and quantitative analyses

The purpose of this study was to determine the subjective and quantitative donor-site morbidity after removal of a free vascularized fibula flap for autoreconstruction. Ten patients and six age-matched, healthy control subjects were included in this study. The postoperative periods ranged from 6 to 87 months. Subjective donor-site morbidity was assessed with a patient questionnaire and the Enneking system. For quantification of donor-site morbidity, gait was evaluated during normal walking, walking under visual and cognitive constraints, and walking at a velocity higher than the preferred one. In general, the patient perception of donor-site morbidity was low. Complaints were frequently mentioned, however, including pain (60 percent), dysesthesia (50 percent), a feeling of ankle instability (30 percent), and inability to run (20 percent). Gait analyses revealed that patients walked at a lower preferred velocity, compared with control subjects. Furthermore, they demonstrated significant increases in the coefficients of variation of stride time during walking under visual and cognitive loads and during walking at a velocity higher than the preferred one, compared with normal walking. These increases were not observed for control subjects. These findings suggest that the reautomatization of gait is affected among patients. This study demonstrates that fibula harvesting is associated with low subjective morbidity but frequent complaints. Walking during complex tasks and at high velocities reveals that restoration of gait is not complete after partial fibulectomy.     

Metal chelates of 2-hydroxy-4-methylthiobutanoic acid in animal feeding: preliminary investigations on stability and bioavailability

The alpha-hydroxyacid 2-hydroxy-4-methylthiobutanoic acid (the so-called methionine hydroxy-analogue, MHA), largely used in animal nutrition as a source of methionine, forms stable metal chelates with divalent metals of formula [[CH(3)SCH(2)CH(2)CH(OH)COO](2)M].ZnH(2)O. Protonation and zinc(II) complex formation constants have been determined by pH-metry at 25 degrees C; the ternary system Zn(2+)/MHA/glycine was also studied by pH-metry and the formation constant of the species [ZnLA] was determined [log beta=6.57(11)]. Experiments in vitro with human intestinal CACO-2 cells indicated that the MHA/Fe chelate was taken up by the cells without any apparent toxic effect.     

N,N′-bis-(2,3-methylenedioxyphenyl)urea and N,N′-bis-(3,4-methylenedioxyphenyl)urea interact with auxin in enhancing root formation of M26 apple (Malus pumila Mill.) stem slices

Stem slices cut from micropropagated cuttings of apple rootstock M26 were cultured in the presence of indole-3-butyric acid (IBA) plus N,N-bis-(2,3-methylenedioxyphenyl)urea or N,N-bis-(3,4-methylenedioxyphenyl)urea, to verify if there was an interaction between them in enhancing root formation. The N,N-bis-(methylenedioxyphenyl)ureas were supplemented after, before and in the simultaneous presence of auxin. Our data demonstrate that only the simultaneous presence of auxin and N,N-bis-(methylenedioxyphenyl)ureas in the culture medium enhanced root formation on M26 stem slices. The percentage of rooted slices obtained in the presence of the mixtures was significantly different from that obtained in the presence of low auxin concentration alone (1µM). Moreover both the percentage of rooted slices and the number of roots per slice obtained in these culture conditions was not significantly different to that of the optimal auxinic treatment in which the auxin concentration was threefold higher.