Glucose sensitivity and pH and thermal stabilities of Trichoderma reesei Cel1A (Bgl II) were improved by site-directed mutagenesis of only two amino acid residues (L167W or P172L) at the entrance of the active site. The Cel1A mutant showed high glucose tolerance (50% of inhibitory concentration = 650 mM), glucose stimulation (2.0 fold at 50 mM glucose), and enhanced specific activity (2.4-fold)...

The QuikChange™ site-directed mutagenesis method is popular but imperfect. An improvement by using partially overlapping primers has been reported several times; however, it is incompatible with the proposed mechanism. The QuikChange™ method using complementary primers is proposed to linearly amplify a target plasmid with the products annealing to produce double-stranded DNA molecules with 5'-o...

Delta-toxin produced by Clostridium perfringens types B and C is a β-pore-forming cytotoxin. Here, using site-directed mutagenesis, we identified the amino acid residues that contribute to delta-toxin oligomerization binding. We replaced Lys-43 Ser-109 located in β-sandwich domain Arg-200 rim domain. Substitution of alanine for caused reductions both cytotoxicity oligomerization. However, excha...

LytA is responsible for the autolysis of many Streptococcus species, including pathogens such as S. pneumoniae, S. pseudopneumoniae and S. mitis. However, how this major autolysin achieves full activity remains unknown. Here, the full-length structure of the S. pneumoniae LytA dimer is reported at 2.1 Å resolution. Each subunit has an N-terminal amidase domain and a C-terminal choline-binding d...

In the European Union, the Committee for Advanced Therapies of the European Medicines Agency takes the lead in the scientific assessment for marketing authorization applications for advanced therapy medicinal products, which include gene therapy medicinal products, somatic cell therapy medicinal products, and tissue-engineered products. The Committee for Advanced Therapies also takes the lead i...

Functional analysis of the Plasmodium falciparum genome is restricted because of the limited ability to genetically manipulate this important human pathogen. We have developed an efficient transposon-mediated insertional mutagenesis method much needed for high-throughput functional genomics of malaria parasites. A drug-selectable marker, human dihydrofolate reductase, added to the lepidopteran ...

Gene therapy using integrating retroviral vectors has proven its effectiveness in several clinical trials for the treatment of inherited diseases and cancer. However, vector-mediated adverse events related to insertional mutagenesis were also observed, emphasizing the need for safer therapeutic vectors. Paradoxically, alpharetroviruses, originally discovered as cancer-causing agents, have a mor...

Neurocristopathies form a specific group of rare genetic diseases in which a defect in neural crest cell development is causal. Because of the large number of neural crest cell derivatives, distinct structures/cell types (isolated or in combination) are affected in each neurocristopathy. The most important issues in this research field is that the underlying genetic cause and associated pathoge...

Ferritins are 24-mer proteins which store and detoxify intracellular iron. Mammalian ferritins are made of two subunit types, the H- and L-chains, with different functional specificity. The H-chain has a metal-binding site (the ferroxidase center) which confers ferroxidase activity to the protein and accelerates iron incorporation. In the L-chain the center is substituted by a salt bridge. We p...

The assembly of enzyme components into the cellulosome complex is dictated by the cohesin-dockerin interaction. In a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P., Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000) Proteins 39, 170-177), we provided experimental evidence that four previously predicted dockerin residues play a decisive role in the specificity of...