Determinants of the recognition of enteroviral cloverleaf RNA by coxsackievirus B3 proteinase 3C

The initiation of enteroviral positive-strand RNA synthesis requires the presence of a functional ribonucleoprotein complex containing a cloverleaf-like RNA secondary structure at the 5' end of the viral genome. Other components of the ribonucleoprotein complex are the viral 3CD proteinase (the precursor protein of the 3C proteinase and the 3D polymerase), the viral 3AB protein and the cellular poly(rC)-binding protein 2. For a molecular characterization of the RNA-binding properties of the enteroviral proteinase, the 3C proteinase of coxsackievirus B3 (CVB3) was bacterially expressed and purified. The recombinant protein is proteolytically active and forms a stable complex with in vitro-transcribed cloverleaf RNA of CVB3. The formation of stable complexes is also demonstrated with cloverleaf RNA of poliovirus (PV) 1, the first cloverleaf of bovine enterovirus (BEV) 1, and human rhinovirus (HRV) 2 but not with cloverleaf RNA of HRV14 and the second cloverleaf of BEV1. The apparent dissociation constants of the protein:RNA complexes range from approx. 1.7 to 4.6 microM. An electrophoretic mobility shift assay with subdomain D of the CVB3 cloverleaf demonstrates that this RNA is sufficient to bind the CVB3 3C proteinase. Binding assays using mutated versions of CVB3 and HRV14 cloverleaf RNAs suggest that the presence of structural features rather than a defined sequence motif of loop D are important for 3C proteinase-RNA interaction.     

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.     

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.     

Engineering tolerance and accumulation of lead and cadmium in transgenic plants

We have studied the utility of the yeast protein YCF1, which detoxifies cadmium by transporting it into vacuoles, for the remediation of lead and cadmium contamination. We found that the yeast YCF1-deletion mutant DTY167 was hypersensitive to Pb(II) as compared with wild-type yeast. DTY167 cells overexpressing YCF1 were more resistant to Pb(II) and Cd(II) than were wild-type cells, and accumulated more lead and cadmium. Analysis of transgenic Arabidopsis thaliana plants overexpressing YCF1 showed that YCF1 is functionally active and that the plants have enhanced tolerance of Pb(II) and Cd(II) and accumulated greater amounts of these metals. These results suggest that transgenic plants expressing YCF1 may be useful for phytoremediation of lead and cadmium.