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Bacterial signaling networks control a wide variety of cellular processes including growth, metabolism, and pathogenesis. Bis-(39--59)-cyclic dimeric guanosine monophosphate (cdiGMP) is a secondary signaling nucleotide that controls cellulose synthesis, biofilm formation, motility and virulence in a wide range of Gram-negative bacterial species. CdiGMP is a dynamic molecule that forms different tertiary structures in vitro, including a trans-monomer, cis-monomer, cis-dimer and G-octaplex (G8). Although the monomer and dimer have been shown to be physiologically relevant in modulating protein activity and transcription, the biological effects of the cdiGMP G8 has not yet been described. Here, we have developed a TLC-based assay to detect radiolabeled cdiGMP G8 formation. Utilizing the radiolabeled cdiGMP G8, we have also shown a novel inhibitory interaction between the cdiGMP G8 and HIV-1 reverse transcriptase and that the cdiGMP G8 does not interact with proteins from Pseudomonas aeruginosa known to bind monomeric and dimeric cdiGMP. These results suggest that the radiolabeled cdiGMP G8 can be used to measure interactions between the cdiGMP G8 and cellular proteins, providing an avenue through which the biological significance of this molecule could be investigated. [ABSTRACT FROM AUTHOR]

Purpose: AAV-mediated gene therapy in the rd10 mouse, with retinal degeneration caused by mutation in the rod cyclic guanosine monophosphate phosphodiesterase b-subunit (PDEb) gene, produces significant, but transient, rescue of photoreceptor structure and function. This study evaluates the ability of AAV-mediated delivery of X-linked inhibitor of apoptosis (XIAP) to enhance and prolong the efficacy of PDEb gene-replacement therapy. Methods: Rd10 mice were bred and housed in darkness. Two groups of animals were generated: Group 1 received subretinal AAV5-XIAP or AAV5-GFP at postnatal age (P) 4 or 21 days; Group 2 received sub-retinal AAV5-XIAP plus AAV5- PDEb, AAV5-GFP plus AAV5- PDEb, or AAV- PDEb alone at age P4 or P21. Animals were maintained for an additional 4 weeks in darkness before being moved to a cyclic-light environment. A subset of animals from Group 1 received a second sub-retinal injection of AAV8-733-PDEb two weeks after being moved to the light. Histology, immunohistochemistry, Western blots, and electroretinograms were performed at different times after moving to the light. Results: Injection of AAV5-XIAP alone at P4 and 21 resulted in significant slowing of light-induced retinal degeneration, as measured by outer nuclear thickness and cell counts, but did not result in improved outer segment structure and rhodopsin localization. In contrast, co-injection of AAV5-XIAP and AAV5-PDEb resulted in increased levels of rescue and decreased rates of retinal degeneration compared to treatment with AAV5-PDEb alone. Mice treated with AAV5-XIAP at P4, but not P21, remained responsive to subsequent rescue by AAV8-733-PDEb when injected two weeks after moving to a light-cycling environment. Conclusions: Adjunctive treatment with the anti-apoptotic gene XIAP confers additive protective effect to genereplacement therapy with AAV5-PDEb in the rd10 mouse. In addition, AAV5-XIAP, when given early, can increase the age at which gene-replacement therapy remains effective, thus effectively prolonging the window of opportunity for therapeutic intervention. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the cardiovascular and renal activities of a newly designed natriuretic peptide (NP). Here, we engineered a novel 28-amino acid chimeric peptide, termed AC-NP that combined the 17-amino acid ring of C type natriuretic peptide (CNP) with the 6-amino acid N-terminus and 5-amino acid C-terminus of atrial natriuretic peptide (ANP). Both in vitro and in vivo experiments were performed to determine the actions of AC-NP. In normal rats, AC-NP proved to be more potentially diuretic, natriuretic and hypotensive compared with other NPs, such as ANP, CNP and vasonatrin peptide (VNP), which is another man-made NP. In relaxation of isolated abdominal aorta from rat, AC-NP was equally effective to ANP, CNP and VNP. Elevated levels of 39,59-guanosine monophosphate (cGMP) in plasma and urine cGMP excretion indicated the participation of cGMP in the functions of AC-NP. Taken together, innovative designed AD-NP might be a new candidate therapeutic peptide against cardiorenal disorders. [ABSTRACT FROM AUTHOR]

The detection of cytosolic DNA, derived from pathogens or host cells, by cytosolic receptors is essential for appropriate host immune responses. Cyclic GMP-AMP synthase (cGAS) is a newly identified cytosolic DNA receptor that produces cyclic GMP-AMP, which activates stimulator of interferon genes (STING), resulting in TBK1-IRF3 pathway activation followed by the production of type I interferons. Here we report the crystal structure of human cGAS. The structure revealed that a cluster of lysine and arginine residues forms the positively charged DNA binding surface of human cGAS, which is important for the STING-dependent immune activation. A structural comparison with other previously determined cGASs and our functional analyses suggested that a conserved zinc finger motif and a leucine residue on the DNA binding surface are crucial for the DNA-specific immune response of human cGAS, consistent with previous work. These structural features properly orient the DNA binding to cGAS, which is critical for DNA-induced cGAS activation and STING-dependent immune activation. Furthermore, we showed that the cGAS-induced activation of STING also involves the activation of the NF-κB and IRF3 pathways. Our results indicated that cGAS is a DNA sensor that efficiently activates the host immune system by inducing two distinct pathways. [ABSTRACT FROM AUTHOR]