Salt-soluble elastin from lathyritic chicks.
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ABSTRACT:
SUBMITTER: Sykes BC
PROVIDER: S-EPMC1168111 | biostudies-other | 1974 Aug
REPOSITORIES: biostudies-other
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Project description:This paper describes the isolation and amino acid analysis of un-cross-linked elastin obtained by neutral salt extraction from the ligamentum nuchae of a calf fed from birth to 9 months on a diet low in copper.
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Project description:BackgroundElastin-like polypeptides (ELPs) are a fascinating biomaterial that has undergone copious development for a variety of therapeutic applications including as a nanoscale drug delivery vehicle. A comprehensive understanding of ELP self-assembly is lacking and this knowledge gap impedes the advancement of ELP-based biomaterials into the clinical realm. The systematic examination of leucine-containing ELPs endeavors to expand existing knowledge about fundamental assembly-disassembly behaviours.ResultsIt was observed that these marginally soluble, short ELPs tend to behave consistently with previous observations related to assembly-related ELP phase transitions but deviated in their disassembly. It was found that chain length, concentration and overall sequence hydrophobicity may influence the irreversible formation of sub-micron particles as well as the formation of multi-micron scale, colloidally unstable aggregates. Amino acid composition affected surface charge and packing density of the particles. Particle stability upon dilution was found to vary depending upon chain length and hydrophobicity, with particles composed of longer and/or more hydrophobic ELPs being more resistant to disassembly upon isothermal dilution.ConclusionsTaken together, these results suggest marginally soluble ELPs may self-assemble but not disassemble as expected and that parameters including particle size, zeta potential and dilution resistance would benefit from widespread systematic evaluations. This information has the potential to reveal novel preparation methods capable of expanding the utility of all existing ELP-based biomaterials.
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Project description:UnlabelledAnion exchanger 2 (AE2), the principal bicarbonate secretor in the human biliary tree, is down-regulated in primary biliary cholangitis. AE2 creates a "bicarbonate umbrella" that protects cholangiocytes from the proapoptotic effects of bile salts by maintaining them deprotonated. We observed that knockdown of AE2 sensitized immortalized H69 human cholangiocytes to not only bile salt-induced apoptosis (BSIA) but also etoposide-induced apoptosis. Because the toxicity of etoposide is pH-independent, there could be a more general mechanism for sensitization of AE2-depleted cholangiocytes to apoptotic stimuli. We found that AE2 deficiency led to intracellular bicarbonate accumulation and increased expression and activity of soluble adenylyl cyclase (sAC), an evolutionarily conserved bicarbonate sensor. Thus, we hypothesized that sAC regulates BSIA. H69 cholangiocytes and primary mouse cholangiocytes were used as models. The sAC-specific inhibitor KH7 not only reversed sensitization to BSIA in AE2-depleted H69 cholangiocytes but even completely prevented BSIA. sAC knockdown by tetracycline-inducible short hairpin RNA also prevented BSIA. In addition, sAC inhibition reversed BSIA membrane blebbing, nuclear condensation, and DNA fragmentation. Furthermore, sAC inhibition also prevented BSIA in primary mouse cholangiocytes. Mechanistically, sAC inhibition prevented Bax phosphorylation at Thr167 and mitochondrial translocation of Bax and cytochrome c release but not c-Jun N-terminal kinase activation during BSIA. Finally, BSIA in H69 cholangiocytes was inhibited by intracellular Ca(2+) chelation, aggravated by thapsigargin, and unaffected by removal of extracellular calcium.ConclusionsBSIA is regulated by sAC, depends on intracellular Ca(2+) stores, and is mediated by the intrinsic apoptotic pathway; down-regulation of AE2 in primary biliary cholangitis sensitizes cholangiocytes to apoptotic insults by activating sAC, which may play a crucial role in disease pathogenesis. (Hepatology 2016;64:522-534).
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