12 research outputs found
A C-terminal cysteine residue is required for peptide-based inhibition of the NGF/TrkA interaction at nM concentrations:implications for peptide-based analgesics
Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor
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Induction of aquaporin 1 but not aquaporin 4 messenger RNA in rat primary brain microvessel endothelial cells in culture
Aquaporins (AQPs) are a family of proteins that mediate water transport across cells, but the extent to which they are involved in water transport across endothelial cells of the blood-brain barrier is not clear. Expression of AQP1 and AQP4 in rat brain microvessel endothelial cells was investigated in order to determine whether these isoforms were present and, in particular, to examine the hypothesis that brain endothelial expression of AQPs is dynamic and regulated by astrocytic influences. Reverse-transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry showed that AQP1 mRNA and protein are present at very low levels in primary rat brain microvessel endothelial cells, and are up-regulated in passaged cells. Upon passage, endothelial cell expression of mdr1a mRNA is decreased, indicating loss of blood-brain barrier phenotype. In passage 4 endothelial cells, AQP1 mRNA levels are reduced by coculture above rat astrocytes, demonstrating that astrocytic influences are important in maintaining the low levels of AQP1 characteristic of the blood-brain barrier endothelium. Reverse-transcriptase-PCR revealed very low levels of AQP1 mRNA present in the RBE4 rat brain microvessel endothelial cell line, with no expression detected in primary cultures of rat astrocytes or in the C6 rat glioma cell line. In contrast, AQP4 mRNA is strongly expressed in astrocytes, but no expression is found in primary or passaged brain microvessel endothelial cells, or in RBE4 or C6 cells. Our results support the concept that expression of AQP1, which is seen in many non-brain endothelia, is suppressed in the specialized endothelium of the blood-brain barrier