7 research outputs found
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Structure-function analysis of connexin32
Individual cell-cell channels consist of two hemichannels, located on neighboring cell membranes, that are interconnected to form an hydrophilic pathway. Each hemichannel, or connexon, is made of six protein subunits, called connexins.Connexin32 liver gap junction protein has four transmembrane segments, two extracellular regions and three cytoplasmic segments, which include the amino and carboxyl termini.The process of cell-cell channel formation was investigated by altering specific amino acids in the presumed extracellular domains of the connexin32. It is these domains that must interact when two hemichannels dock to form an open cell-cell channel. The mutant connexins were generated by site-directed in vitro mutagenesis of a connexin32 cDNA. The mutated cDNAs were then transcribed in vitro and the mRNA was injected into Xenopus oocytes for expression. Junctional conductances between paired oocytes resulting from the expression of the mRNA were measured by the double-voltage clamp technique.Every amino acid replacement in connexin32 affected its channel-forming ability. Some replacements altered the docking specificity of the hemichannel. The replacement of any one of the six cysteines in the extracellular loops by serine yielded proteins incapable of making channels, indicating that the extracellular cysteines play a crucial role in channel formation.In addition, deletion mutants in the carboxyterminal domain of connexin32 were generated in order to determine the shortest segment still capable of forming channels. The transmembrane topology of some of these deletion mutants was investigated by in vitro translation in a reticulocyte lysate supplemented with canine microsomes.Net positive charge and length of the carboxyterminal segment were found to be determinants for channel formation. Phosphorylation of Ser233 and Ser240 was found not to be necessary for channel formation when expressed in Xenopus oocytes.Finally, connexin32 hexamer formation was studied by native polyacrylamide gel electrophoresis, sucrose gradients of the in vitro translation products combined with crosslinking reagents
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Cell/cell channel formation involves disulfide exchange
The oocyte cell/cellâchannel assay was used to identify amino acids involved in the process of cell/cellâchannel formation. The expression of the rat liver gapâjunction protein, connexin 32, in single oocytes, results in the accumulation of a pool of channel precursors. Upon pairing of such oocytes, cell/cell channels form rapidly from this pool. The rate of formation is affected by thiolâspecific reagents and the pH. This suggests the involvement of extracellular cysteine residues in the channel formation process. Two connexinâ32 mutants were generated by siteâdirected mutagenesis in which cysteine residues were replaced by serine. Both mutant connexins were unable to form cell/cell channels. Thus, the cysteine residues appear to play an important role in the channel formation process
A connexin-32 mutation associated with Charcot-Marie-Tooth disease does not affect channel formation in oocytes
AbstractMembers of the connexin family differ most in their carboxy-termini, both with respect to sequence and length. In order to assess the contribution of this region to channel function, a series of carboxy-terminal deletion mutants were tested in the paired-oocyte expression system. Connexin-32 can be truncated by 64 amino acids without detectable loss of its known channel properties. Removal of additional amino acids results in a progressive loss of function over a stretch of 4 amino acids. In addition to this effect of length the charge of the carboxy-terminus appears to be another determinant of channel function. One of the fully functional deletion mutants, carrying a stop codon after amino acid-219, had been reported to be associated with Charcot-Marie-Tooth disease. The implications of this finding are discussed
Perspectives on Strengthening Cancer Research and Control in Latin America Through Partnerships and Diplomacy: Experience of the National Cancer Instituteâs Center for Global Health
According to the Pan American Health Organization, noncommunicable diseases, including cancer, are the leading causes of preventable and premature death in the Americas. Governments and health care systems in Latin America face numerous challenges as a result of increasing morbidity and mortality from cancer. Multiple international organizations have recognized the need for collaborative action on and technical support for cancer research and control in Latin America. The Center for Global Health at the US National Cancer Institute (NCI-CGH) is one entity among many that are working in the region and has sought to develop a strategy for working in Latin America that draws on and expands the collaborative potential of engaged, skilled, and diverse partners. NCI-CGH has worked toward developing and implementing initiatives in collaboration with global partners that share the common objectives of building a global cancer research community and translating research results into evidence-informed policy and practice. Both objectives are complementary and synergistic and are additionally supported by an overarching strategic framework that is focused on partnerships and science diplomacy. This work highlights the overall strategy for NCI-CGH engagement in Latin America through partnerships and diplomacy, and highlights selected collaborative efforts that are aimed at improving cancer outcomes in the region