AIM: To verify whether 'defective' mutations existed in hepatitis D virus (HDV).METHODS: Hepatitis delta antigen (HDAg)-codingsequences were amplified using Pfu DNA polymerases with proof-reading activities fr...AIM: To verify whether 'defective' mutations existed in hepatitis D virus (HDV).METHODS: Hepatitis delta antigen (HDAg)-codingsequences were amplified using Pfu DNA polymerases with proof-reading activities from sera of five patients with chronic hepatitis D. Multiple colonies were sequenced for each patient. Pfu analyzed a total of 270 HDV clones.Three representative defective HDV clones were constructed in expression plasmids and transfected into a human hepatoma cell line. Cellular proteins were extracted and analyzed by Western blot.RESULTS: Four of five cases (80%) showed defective HDV genomes in their sera. The percentage of defective genomes was 3.7% (10/270). The majority (90%) of the defective mutations were insertions or deletions that resulted in frameshift and abnormal stop translation of the HDAg. The predicted mutated HDAg ranged from 45amino acids to ＞214 amino acids in length. Various domains of HDAg associated with viral replication or packaging were affected in different HDV isolates. Western blot analysis showed defected HDAg in predicted positions.CONCLUSION: 'Defective' viruses do exist in chronic HDV infected patients, but represented as minor strains. The clinical significance of the 'defected' HDV needs further study to evaluate.展开更多
Fluorescence lifetime imaging microscopy （FLIM） has gained popularity as a sensitive technique to monitor the functional/conformational states of reduced nicotinamide adenine dinucleotide （NADH）, one of the main c...Fluorescence lifetime imaging microscopy （FLIM） has gained popularity as a sensitive technique to monitor the functional/conformational states of reduced nicotinamide adenine dinucleotide （NADH）, one of the main compounds of oxidative phosphorylation. In this letter, we apply the technique to characterize the metabolic changes in mouse embryonic fibroblast 3T3 cells upon bacterial infection. A gradual shortening of the decaying time constants in both the short and the long lifetime components of NADH＇s autofluorescence is detected. The ratio of the short and the long lifetime components＇ relative contributions, however, shows a rapid increase, indicating the rise of cellular metabolic activity over the course of infection.展开更多
基金Supported by grants from the National Science Council (NSC90-2314-B-010-016, NSC91-2314-B-010-080-MH), Taiwan, China
文摘AIM: To verify whether 'defective' mutations existed in hepatitis D virus (HDV).METHODS: Hepatitis delta antigen (HDAg)-codingsequences were amplified using Pfu DNA polymerases with proof-reading activities from sera of five patients with chronic hepatitis D. Multiple colonies were sequenced for each patient. Pfu analyzed a total of 270 HDV clones.Three representative defective HDV clones were constructed in expression plasmids and transfected into a human hepatoma cell line. Cellular proteins were extracted and analyzed by Western blot.RESULTS: Four of five cases (80%) showed defective HDV genomes in their sera. The percentage of defective genomes was 3.7% (10/270). The majority (90%) of the defective mutations were insertions or deletions that resulted in frameshift and abnormal stop translation of the HDAg. The predicted mutated HDAg ranged from 45amino acids to ＞214 amino acids in length. Various domains of HDAg associated with viral replication or packaging were affected in different HDV isolates. Western blot analysis showed defected HDAg in predicted positions.CONCLUSION: 'Defective' viruses do exist in chronic HDV infected patients, but represented as minor strains. The clinical significance of the 'defected' HDV needs further study to evaluate.
基金support from the National Science Council (NSC 97-3112-B-010-006, NSC96-2112-M-010-001, and NSC98-2112-M-010-001-MY3)the Ministry of Education (Aim for Top University Project)
文摘Fluorescence lifetime imaging microscopy （FLIM） has gained popularity as a sensitive technique to monitor the functional/conformational states of reduced nicotinamide adenine dinucleotide （NADH）, one of the main compounds of oxidative phosphorylation. In this letter, we apply the technique to characterize the metabolic changes in mouse embryonic fibroblast 3T3 cells upon bacterial infection. A gradual shortening of the decaying time constants in both the short and the long lifetime components of NADH＇s autofluorescence is detected. The ratio of the short and the long lifetime components＇ relative contributions, however, shows a rapid increase, indicating the rise of cellular metabolic activity over the course of infection.
