Poxvirus uracil‐DNA glycosylase—An unusual member of the family I uracil‐DNA glycosylases

Uracil‐DNA glycosylases are ubiquitous enzymes, which play a key role repairing damages in DNA and in maintaining genomic integrity by catalyzing the first step in the base excision repair pathway. Within the superfamily of uracil‐DNA glycosylases family I enzymes or UNGs are specific for recognizing and removing uracil from DNA. These enzymes feature conserved structural folds, active site residues and use common motifs for DNA binding, uracil recognition and catalysis. Within this family the enzymes of poxviruses are unique and most remarkable in terms of amino acid sequences, characteristic motifs and more importantly for their novel non‐enzymatic function in DNA replication. UNG of vaccinia virus, also known as D4, is the most extensively characterized UNG of the poxvirus family. D4 forms an unusual heterodimeric processivity factor by attaching to a poxvirus‐specific protein A20, which also binds to the DNA polymerase E9 and recruits other proteins necessary for replication. D4 is thus integrated in the DNA polymerase complex, and its DNA‐binding and DNA scanning abilities couple DNA processivity and DNA base excision repair at the replication fork. The adaptations necessary for taking on the new function are reflected in the amino acid sequence and the three‐dimensional structure of D4. An overview of the current state of the knowledge on the structure‐function relationship of D4 is provided here.     

Infrared Thermography and Ultrasonography to Indirectly Monitor the Influence of Liner Type and Overmilking on Teat Tissue Recovery

Eight Danish Holstein cows were milked with a 1-mm thick specially designed soft liner on their right rear teat and a standard liner mounted under extra high tension on their left rear teat. Four of the animals were overmilked for 5 min. Rear teats were subjected to ultrasound examination on the first day and to infrared thermography on the second day. Teats were submersed in ethanol 20 min post-milking on the second day. Ultrasonography measurements showed that teat canal length increased by 30–41% during milking. Twenty minutes after milking, teats milked with modified standard liners still had elongated teat canals while teats milked with the soft liner were normalized. Overmilking tended to increase teat wall thickness. Approximately 80% of variability in teat canal length, from before teat preparation to after milking, could be explained by changes during teat preparation. Thermography indicated a general drop in teat temperature during teat preparation. Teat temperature increased during milking and continued to increase until the ethanol challenge induced a significant drop. Temperatures approached pre-challenge rather than pre-milking temperatures within 10 minutes after challenge. Teat temperatures were dependent on type of liner. Mid-teat temperatures post-challenge relative to pre-teat preparation were dependent on overmilking. Thermography and ultrasound were considered useful methods to indirectly and non invasively evaluate teat tissue integrity.     

重组腺相关病毒2型转导人外周血单核细胞来源树突状细胞的研究

To find out the infection efficiency of recombinant adeno-as sociated virus 2-mediated exogenou s genes in human peripheral blood monocyte-derived dendritic cells(D Cs),the process of transfection wa s investigated by FITC-labeled rAA V2,and observed under confocal mic roscope.Newly separated dendritic cells were tranfected by rAAV2-luc and rAAV2-GFP at different MOI,and transfection efficiency were detec ted by luminometer for rAAV2-luc a nd flow cytometry for rAAV2-GFP.Th e results were elucidated in four different assay systems:(1)60min w as needed for rAAV2 to bind on den dritic cells,and got into cells in the following 10min;(2)the express ion of luc could be detected at th e MOI as low as 1×10~5v.g/cell,and the expression plateau was reached by the MOI of 10~6～10~7v.g/cell,furt her increase of MOI had no functio n on expression level;(3)transgene expression was detected after 48h, and maintained a higher expression level from 96h to 240h after infec tion;(4)7days postinfection of rAA V2-GFP,5%～18% dendritic cells were GFP positive. These data suggest th at rAAV2 vector can efficiently in fect monocyte-derived dendritic ce lls and mediate exogenous gene exp ression,and that the application o f rAAV2 as vector may be useful fo r gene transfer to dendritic cells in ex vivo immunotherapy.     

Evolution of body condition‐dependent dispersal in metapopulations

Body condition‐dependent dispersal strategies are common in nature. Although it is obvious that environmental constraints may induce a positive relationship between body condition and dispersal, it is not clear whether positive body conditional dispersal strategies may evolve as a strategy in metapopulations. We have developed an individual‐based simulation model to investigate how body condition–dispersal reaction norms evolve in metapopulations that are characterized by different levels of environmental stochasticity and dispersal mortality. In the model, body condition is related to fecundity and determined either by environmental conditions during juvenile development (adult dispersal) or by those experienced by the mother (natal dispersal). Evolutionarily stable reaction norms strongly depend on metapopulation conditions: positive body condition dependency of dispersal evolved in metapopulation conditions with low levels of dispersal mortality and high levels of environmental stochasticity. Negative body condition‐dependent dispersal evolved in metapopulations with high dispersal mortality and low environmental stochasticity. The latter strategy is responsible for higher dispersal rates under kin competition when dispersal decisions are based on body condition reached at the adult life stage. The evolution of both positive and negative body condition‐dependent dispersal strategies is consequently likely in metapopulations and depends on the prevalent environmental conditions.     

Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry

In the double-stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid-bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N-terminal helix-loop-helix (H-L-H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid-bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.     

DNA packaging: a new class of molecular motors

DNA is packaged into preformed bacteriophage capsids to liquid crystalline density by the action of a portal protein complex. Single molecule packaging studies indicate that this is a new and extremely powerful class of molecular motors.     

Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro

Coat and scaffolding subunits derived from P22 procapsids have been purified in forms that co-assemble rapidly and efficiently into icosahedral shells in vitro under native conditions. The half-time for this reaction is approximately five minutes at 21 degrees C. The in vitro reaction exhibits the regulated features observed in vivo. Neither coat nor scaffolding subunits alone self-assemble into large structures. Upon mixing the subunits together they polymerize into procapsid-like shells with the in vivo coat and scaffolding protein composition. The subunits in the purified coat protein preparations are monomeric. The scaffolding subunits appear to be monomeric or dimeric. These results confirm that P22 procapsid formation does not proceed through the assembly of a core of scaffolding, which then organizes the coat, but requires copolymerization of coat and scaffolding. To explore the mechanisms of the control of polymerization, shell assembly was examined as a function of the input ratio of scaffolding to coat subunits. The results indicated that scaffolding protein was required for both initiation of shell assembly and continued polymerization. Though procapsids produced in vivo contain about 300 molecules of scaffolding, shells with fewer subunits could be assembled down to a lower limit of about 140 scaffolding subunits per shell. The overall results of these experiments indicate that coat and scaffolding subunits must interact in both the initiation and the growth phases of shell assembly. However, it remains unclear whether during growth the coat and scaffolding subunits form a mixed oligomer prior to adding to the shell or whether this occurs at the growing edge.