Cloning and characterization of new cellulases from Cellulomonas fimi and Cellulomonas flavigena

Lignocellulose is one of the most abundant carbon sources in nature. This naturally-occuring substance is an underutilized source of bioenergy. A major bottleneck in biofuel processing is the enzymatic hydrolysis of lignocellulose into its ultimate fermentable product, glucose. Cellulomonas fimi is a well-studied soil organism known for its capabilities to efficiently hydrolyze cellulose. Recently sequenced genomes of Cellulomonas fimi and Cellulomonas flavigena have allowed analysis to reveal previously unidentified cellulases from several glycoside hydrolase (GH) families. This study also includes the expression of secreted cellulases from families GH 5, 6, and 9 at the protein level by the native organism after growth in media supplemented with carboxymethylcellulose or soluble xylan. In order to find enzymes with novel qualities, the cloning and expression of these newly identified cellulases from C. fimi and C. flavigena were done. One of these enzymes is Celf_1230 (Cel6C), a putative cellobiohydrolase from the glycoside hydrolase family 6. Using substituted cellulose derivatives as substrates, we have characterized Celf_1230 to be a thermostable enzyme with endoglucanase activity

Cortactin depletion results in short tubulobulbar complexes and spermiation failure in rat testes

Summary Tubulobulbar complexes are actin-related endocytic structures that form at sites of intercellular attachment in the seminiferous epithelium and are proposed to internalize intact junctions. In this study, we test the prediction that altering the structure/function of tubulobulbar complexes results in failure to release mature spermatids from Sertoli cells. We used an in vivo knockdown strategy to target cortactin, a component of tubulobulbar complexes, in Sprague Dawley rats. In each animal, one testis was surgically injected with cortactin siRNA reagents and the other testis was injected with non-targeting siRNA. After three days, experimental and control testes were processed for immunoblotting, electron microscopy or immunofluorescence microscopy. In testis sections immunostained for cortactin or labeled for filamentous actin, fluorescence microscopy revealed that tubulobulbar complexes were shorter in siRNA-treated testes relative to controls. Significantly, in the knockdown testes, spermiation was delayed in some tubules and had failed in others. When evaluated by electron microscopy, adhesion complexes (ectoplasmic specializations) remained associated with mature spermatids that failed to be released from Sertoli cells. Immunoblots both of whole testis lysates and of isolated seminiferous epithelial lysates confirmed that cortactin expression was knocked-down in experimental testes and in the seminiferous epithelium respectively, relative to controls. Moreover, in testes injected with siRNA reagents with a dye modification on one of the four targeting siRNA sequences, dye clusters were detected at the base of the epithelium confirming that the reagents entered Sertoli cells. Our results are consistent with the hypothesis that tubulobulbar complexes internalize intercellular junctions and that they are a significant component of the sperm release mechanism