Protein crystals in adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.).

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress

Wireless capsule endoscopy versus ileocolonoscopy for the diagnosis of postoperative recurrence of Crohn's disease: a prospective study

BACKGROUND AND AIMS: Following ileocolonic resection for Crohn's disease (CD), early endoscopic recurrence predicts recurrence of symptoms. The aim of the study was to compare ileocolonoscopy and wireless capsule endoscopy (WCE) for the detection of postoperative recurrence in CD. METHODS: WCE and ileocolonoscopy were performed within six months following surgery in 32 prospectively enrolled patients. Two independent observers interpreted the results of WCE. Recurrence in the neoterminal ileum was defined by a Rutgeerts score ⩾1. When observers at WCE did not concur, WCE results were considered as either true negative or true positive and sensitivity and specificity were calculated according to both assumptions. RESULTS: Recurrence occurred in 21 patients (68%) and was detected by ileocolonoscopy in 19 patients. Sensitivity was 90% and specificity 100%. Sensitivity of WCE was 62% and 76% and specificity was 100% and 90%, respectively, depending on assumptions. There was a correlation between the severity of the lesions measured by both methods (p<0.05). Lesions located outside the scope of conventional endoscopy were detected by WCE in two thirds of patients with excellent interobserver agreement (kappa >0.9) for all lesions with the exception of ulceration (kappa = 0.7). CONCLUSIONS: The sensitivity of WCE in detecting recurrence in the neoterminal ileum was inferior to that of ileocolonoscopy. In contrast, WCE detected lesions outside the scope of ileocolonoscopy in more than two thirds of patients. Additional follow up studies are needed to assess the clinical relevance of such lesions. At the present time, it seems that WCE cannot systematically replace ileocolonoscopy in the regular management of patients after surgery