Gene therapy for pathological scar with hepatocyte growth factor mediated by recombinant adenovirus vector

Pathologic scar, characterized by excessive dermal fibrosis and scarring, is a common im-portant clinical sequela after wound healing. It often appears during wound healing after deep burn, surgical cutting and other injured skin. Accumulation of extracellular matrix (ECM) proteins is a manifestation of increased collagen synthesis and/or reduced matrix degradation, resulting in excessive scarring with a deformed appearance and dysfunction[1]. To date, treatment modalities to scar include sur…     

Nitrite transport is mediated by the nitrite-specific high-affinity NitA transporter and by nitrate transporters NrtA, NrtB in Aspergillus nidulans

Disruption of the Aspergillus nidulans high-affinity nitrate transporter genes (nrtA and nrtB) prevents growth on nitrate but not nitrite. We identified a distinct nitrite transporter (K(m)=4.2+/-1 microM, V(max)=168+/-21 nmolmg(-1)DW(-1)h(-1)), designated NitA. Disruption of nrtA, nrtB and nitA blocked growth on nitrite, despite low rates of nitrite depletion we ascribe to passive nitrous acid permeation. Growth of the single mutant nitA16 on nitrite was wild-type, suggesting that NrtA and/or NrtB transports nitrite as well as nitrate. Indeed, NrtA and NrtB transport nitrite at higher rates than NitA; K(m) and V(max) values were 16+/-4 microM and 808+/-67 nmolmg(-1)DW(-1)h(-1) (NrtA) and 11+/-1 microM and 979+/-17 nmolmg(-1)DW(-1)h(-1) (NrtB). We suggest that NrtA is a nitrate/nitrite transporter, NrtB absorbs nitrite in preference to nitrate and NitA is exclusively a nitrite transporter.     

Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance

Much attention has been paid to the signal sequences of eukaryotic protoporphyrinogen oxidases (protoxes); both the organelles targeted by protoxes and the role of protoxes in conferring resistance against protox‐inhibiting herbicides, such as oxyfluorfen, have been examined. However, there have been no reports on the translocation of prokaryotic protoxes. This study investigated the targeting ability of Myxococcus xanthus protox in vitro and in vivo. In an in vitro translocation assay using a dual import system, M. xanthus protein was detected in chloroplasts and mitochondria, suggesting that the M. xanthus protox protein was targeted into both organelles. In order to confirm the in vitro dual targeting ability of M. xanthus, we used a stable transgenic strategy to investigate dual targeting in vivo. In transgenic rice plants overexpressing M. xanthus protox, M. xanthus protox antibody cross‐reacted with proteins with predicted molecular masses of 50 kDa from both chloroplasts and mitochondria, and this in vivo transgene expression corresponded to a prominent increase in chloroplastic and mitochondrial protox activity. Seeds from the transgenic lines M4 and M7 germinated in solid Murashige and Skoog media of up to 500 µm of oxyfluorfen, whereas wild‐type seeds did not germinate in 1 µm . After 4‐week‐old‐rice plants were treated with oxyfluorfen for 3 d, lines M4 and M7 exhibited normal growth, whereas the wild‐type line was severely bleached and necrotized. The herbicidal resistance is attributed to the insignificant accumulation of photodynamic protoporphyrin IX in cytosol because the high chloroplastic and mitochondrial protox activity in oxyfluorfen‐treated transgenic lines, compared with that in oxyfluorfen‐treated and untreated wild‐type plants, metabolizes protoporphyrinogen IX to chlorophyll and heme. A practical application of the dual targeting of M. xanthus protox for obtaining outstanding resistance to peroxidizing herbicides is discussed.     

The yeast recombinational repair protein Rad59 interacts with Rad52 and stimulates single-strand annealing

The yeast RAD52 gene is essential for homology-dependent repair of DNA double-strand breaks. In vitro, Rad52 binds to single- and double-stranded DNA and promotes annealing of complementary single-stranded DNA. Genetic studies indicate that the Rad52 and Rad59 proteins act in the same recombination pathway either as a complex or through overlapping functions. Here we demonstrate physical interaction between Rad52 and Rad59 using the yeast two-hybrid system and co-immunoprecipitation from yeast extracts. Purified Rad59 efficiently anneals complementary oligonucleotides and is able to overcome the inhibition to annealing imposed by replication protein A (RPA). Although Rad59 has strand-annealing activity by itself in vitro, this activity is insufficient to promote strand annealing in vivo in the absence of Rad52. The rfa1-D288Y allele partially suppresses the in vivo strand-annealing defect of rad52 mutants, but this is independent of RAD59. These results suggest that in vivo Rad59 is unable to compete with RPA for single-stranded DNA and therefore is unable to promote single-strand annealing. Instead, Rad59 appears to augment the activity of Rad52 in strand annealing.     

The Rad52-Rad59 complex interacts with Rad51 and replication protein A

The RAD52 gene is essential for homology-dependent repair of double-strand breaks in Saccharomyces cerevisiae. Rad52 forms complexes with Rad51, replication protein A (RPA) or Rad59 and its presence is essential for the formation of Rad51-Rad52-Rad59 and RPA-Rad52-Rad59 complexes. The N-terminal region of Rad52, which is required for self-interaction to form a ring structure, is required for interaction with Rad59. Rad59 also shows self-interaction suggesting the formation of heteromeric and homomeric rings of Rad52 and Rad59. In wild-type cells, we propose the Rad51-Rad52-Rad59 complex is involved in conservative recombination events, including gene conversion and reciprocal recombination, whereas the Rad52-Rad59 complex participates in single-strand annealing.     

RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates

DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system. RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.     

Evidence for extensive gene flow and Thermotoga subpopulations in subsurface and marine environments

Oil reservoirs represent a nutrient-rich ecological niche of the deep biosphere. Although most oil reservoirs are occupied by microbial populations, when and how the microbes colonized these environments remains unanswered. To address this question, we compared 11 genomes of Thermotoga maritima-like hyperthermophilic bacteria from two environment types: subsurface oil reservoirs in the North Sea and Japan, and marine sites located in the Kuril Islands, Italy and the Azores. We complemented our genomes with Thermotoga DNA from publicly available subsurface metagenomes from North America and Australia. Our analysis revealed complex non-bifurcating evolutionary history of the isolates'' genomes, suggesting high amounts of gene flow across all sampled locations, a conjecture supported by numerous recombination events. Genomes from the same type of environment tend to be more similar, and have exchanged more genes with each other than with geographically close isolates from different types of environments. Hence, Thermotoga populations of oil reservoirs do not appear isolated, a requirement of the ‘burial and isolation'' hypothesis, under which reservoir bacteria are descendants of the isolated communities buried with sediments that over time became oil reservoirs. Instead, our analysis supports a more complex view, where bacteria from subsurface and marine populations have been continuously migrating into the oil reservoirs and influencing their genetic composition. The Thermotoga spp. in the oil reservoirs in the North Sea and Japan probably entered the reservoirs shortly after they were formed. An Australian oil reservoir, on the other hand, was likely colonized very recently, perhaps during human reservoir development.     

基因工程改善植物生物质能利用的研究进展

利用植物木质纤维资源发酵产乙醇越来越受到人们的重视,但是要达到工业生产仍然存在很多难题。最近在利用植物基因工程技术改善植物自身性状,以利于能源植物的研究方面取得了一定的进展,这些研究包括减少植物自身细胞壁中的木质素含量、细胞中积累表达纤维素酶和木聚耱酶等的方法,使产生的生物质更利于降解利用。