PubMed · 7531960
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K S Saini, B Bhandari. 1994. An improved method to do dot blot analysis.. https://doi.org/10.1006/abio.1994.1491
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Obesity is an increasing pathophysiological problem in developed societies. Despite all major progress in understanding molecular mechanisms of obesity, currently available anti-obesity drugs have shown limited efficacy with severe side effects. CRISPR interference (CRISPRi) mechanism based on catalytically dead Cas9 (dCas9) and single guide RNA (sgRNA) was combined with a targeted nonviral gene delivery system to treat obesity and obesity-induced type 2 diabetes. A fusion peptide targeting a vascular and cellular marker of adipose tissue, prohibitin, was developed by conjugation of adipocyte targeting sequence (CKGGRAKDC) to 9-mer arginine (ATS-9R). (dCas9/sgFabp4) + ATS-9R oligoplexes showed effective condensation and selective delivery into mature adipocytes. Targeted delivery of the CRISPRi system against Fabp4 to white adipocytes by ATS-9R induced effective silencing of Fabp4, resulting in reduction of body weight and inflammation and restoration of hepatic steatosis in obese mice. This RNA-guided DNA recognition platform provides a simple and safe approach to regress and treat obesity and obesity-induced metabolic syndromes.
Vasodilator-stimulated phosphoprotein (VASP) is involved in multiple actin-mediated processes, including regulation of serum response factor (SRF) activity. We used the SRF transcriptional assay to define functional domains in VASP and to show that they coincide with those required for F-actin accumulation, as determined by a quantitative FACS assay. We identified inactive VASP mutants that can interfere both with F-actin assembly and with SRF activation by wild-type VASP. These VASP mutants also inhibit actin-based motility of Vaccinia virus and Shigella flexneri. VASP-induced F-actin accumulation and SRF activation require both functional Rho and its effector mDia, and conversely, mDia-mediated SRF activation is critically dependent on functional VASP. VASP and mDia also associate physically in vivo. These findings show that VASP and mDia function cooperatively downstream of Rho to control F-actin assembly and SRF activity.
The oncoprotein ErbB2 is frequently overexpressed in human tumours, but no activating ErbB2-specific ligand has yet been identified. Here we analyse the catalytic and oligomeric behaviour of ErbB2 using phosphorylation-state-specific antibodies which distinguish kinase-active and -inactive ErbB2 receptor subsets. Heregulin-alpha (HRG) activates ErbB2 in G8/DHFR 3T3 cells by selectively inducing hetero-oligomerization with kinase-defective ErbB3, indicating that heterologous transphosphorylation is an unlikely prerequisite for ErbB2 activation. HRG also triggers association of epidermal-growth-factor receptors (EGFR) with a kinase-inactive ErbB2 subset while reducing EGFR association with active ErbB2. Similarly, EGF treatment of A431 cells induces concomitant hetero-oligomerization of active ErbB2 with inactive EGFR, of active EGFR with inactive ErbB2, and of inactive ErbB2 with kinase-defective ErbB3. These combinatorial patterns of ligand-dependent oligomerization suggest a multivalent model of receptor tyrosine kinase interaction in which liganded homodimers provide stable oligomerization interfaces for unliganded ErbB2 or other bystander receptors. We submit that ErbB2 may be physiologically activated via a 'proxy' ligand-inducible heterotetrameric mechanism similar to that already established for transforming-growth-factor-beta type I receptors.