Intergeneric fusant development using chitinase preparation of Rhizopus stolonifer NCIM 880.

Fungal chitinase have tremendous applications in biotech industries, with this approach we focused on extracellular chitinase from Rhizopus stolonifer NCIM 880 for the formation of fungal protoplasts. The maximum chitinase production reached after 24 h at 2.5% colloidal chitin concentration in presence of starch as an inducer. Chitinase was extracted efficiently at 65% cold acetone concentration and then purified by using DEAE-Cellulose column chromatography. Purified chitinase having molecular weight 22 kDa with single polypeptide chain was optimally active at pH 5.0 and temperature 30 °C. The purified chitinase revealed kinetic properties like Km 1.66 mg/ml and Vmax 769 mM/min. Crude chitinase extract efficiently formed protoplasts from A. niger, A. oryzae, T. viride and F. moniliforme. The formed protoplasts of A. niger and T. viride showed 70 and 66% regeneration frequency respectively. Further, intergeneric fusants were developed successfully and identified at molecular level using RNA profiling. Thus, this study could be useful for strain improvement of various fungi for biotechnological applications.

MUC1 extracellular domain confers resistance of epithelial cancer cells to anoikis.

Anoikis, a special apoptotic process occurring in response to loss of cell adhesion to the extracellular matrix, is a fundamental surveillance process for maintaining tissue homeostasis. Resistance to anoikis characterises cancer cells and is a pre-requisite for metastasis. This study shows that overexpression of the transmembrane mucin protein MUC1 prevents initiation of anoikis in epithelial cancer cells in response to loss of adhesion. We show that this effect is largely attributed to the elongated and heavily glycosylated extracellular domain of MUC1 that protrudes high above the cell membrane and hence prevents activation of the cell surface anoikis-initiating molecules such as integrins and death receptors by providing them a mechanically 'homing' microenvironment. As overexpression of MUC1 is a common feature of epithelial cancers and as resistance to anoikis is a hallmark of both oncogenic epithelial-mesenchymal transition and metastasis, MUC1-mediated cell resistance to anoikis may represent one of the fundamental regulatory mechanisms in tumourigenesis and metastasis.

Raf1 Is a DCAF for the Rik1 DDB1-like protein and has separable roles in siRNA generation and chromatin modification.

Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.