Generation of Infectious Mimivirus Virions Through Inoculation of Viral DNA Within Acanthamoeba castellanii Shows Involvement of Five Proteins, Essentially Uncharacterized.

One of the most curious findings associated with the discovery of Acanthamoeba polyphaga mimivirus (APMV) was the presence of many proteins and RNAs within the virion. Although some hypotheses on their role in Acanthamoeba infection have been put forward, none have been validated. In this study, we directly transfected mimivirus DNA with or without additional proteinase K treatment to extracted DNA into Acanthamoeba castellanii. In this way, it was possible to generate infectious APMV virions, but only without extra proteinase K treatment of extracted DNA. The virus genomes before and after transfection were identical. We searched for the remaining DNA-associated proteins that were digested by proteinase K and could visualize at least five putative proteins. Matrix-assisted laser desorption/ionization time-of-flight and liquid chromatography-mass spectrometry comparison with protein databases allowed the identification of four hypothetical proteins-L442, L724, L829, and R387-and putative GMC-type oxidoreductase R135. We believe that L442 plays a major role in this protein-DNA interaction. In the future, expression in vectors and then diffraction of X-rays by protein crystals could help reveal the exact structure of this protein and its precise role.

Rapid Diagnosis of Lung Tumors, a Feasability Study Using Maldi-Tof Mass Spectrometry.

OBJECTIVE: Despite recent advances in imaging and core or endoscopic biopsies, a percentage of patients have a major lung resection without diagnosis. We aimed to assess the feasibility of a rapid tissue preparation/analysis to discriminate cancerous from non-cancerous lung tissue. METHODS: Fresh sample preparations were analyzed with the Microflex LTTM MALDI-TOF analyzer. Each main reference spectra (MSP) was consecutively included in a database. After definitive pathological diagnosis, each MSP was labeled as either cancerous or non-cancerous (normal, inflammatory, infectious nodules). A strategy was constructed based on the number of concordant responses of a mass spectrometry scoring algorithm. A 3-step evaluation included an internal and blind validation of a preliminary database (n = 182 reference spectra from the 100 first patients), followed by validation on a whole cohort database (n = 300 reference spectra from 159 patients). Diagnostic performance indicators were calculated. RESULTS: 127 cancerous and 173 non-cancerous samples (144 peripheral biopsies and 29 inflammatory or infectious lesions) were processed within 30 minutes after biopsy sampling. At the most discriminatory level, the samples were correctly classified with a sensitivity, specificity and global accuracy of 92.1%, 97.1% and 95%, respectively. CONCLUSIONS: The feasibility of rapid MALDI-TOF analysis, coupled with a very simple lung preparation procedure, appears promising and should be tested in several surgical settings where rapid on-site evaluation of abnormal tissue is required. In the operating room, it appears promising in case of tumors with an uncertain preoperative diagnosis and should be tested as a complementary approach to frozen-biopsy analysis.

Oligomeric states of the detergent-solubilized human serum paraoxonase (PON1).

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C(12)E(8) micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.