Regulation of mammalian photoreceptor phosphodiesterase (PDE) by its noncatalytic GAF domains and its two small subunits, gamma and delta

cGMP phosphodiesterase (PDE) is the central effector of visual transduction in retinal rod photoreceptors. The overall goal of this work was to investigate the possible regulatory mechanisms of PDE in mammalian rod photoreceptors by its noncatalytic cGMP binding sites on the PDE catalytic dimer (Palphabeta), and by the interactions between the inhibitory Pgamma subunit and Palphabeta in their phosphorylated and non-phosphorylated states. First, the binding of cGMP to the noncatalytic sites on membrane-associated PDE (PDE-M) and soluble PDE (PDE-S) was characterized. We found that PDE-M holoenzyme contains 2 non-identical high-affinity cGMP binding sites; one of the cGMP binding sites is functionally nonexchangeable. In contrast, PDE-S can release and bind cGMP at both noncatalytic sites. Activation of PDE reduces more than 100-fold its cGMP binding affinity at one site, while the second cGMP binding site exhibits a 3-fold reduction in binding affinity. We postulate that cGMP dissociation from these two non-identical binding sites might contribute differently to controlling the activation and inactivation kinetics of PDE6 during visual transduction in rod photoreceptors. We also studied interactions between Pgamma and the mammalian Palphabeta catalytic dimer. It was found that Pgamma binds to two distinct sites on the catalytic Palphabeta dimer. cGMP occupancy at the noncatalytic binding sites is responsible for this binding heterogeneity. Two major domains on Pgamma interact with Palphabeta independently. The N-terminal half of Pgamma functions to restore and stabilize cGMP binding at the GAF domain with a binding affinity 50-fold greater than its C-terminal, inhibitory region. Phosphorylation is an important regulatory mechanism in the visual transduction pathway. Our results showed that both Pgamma and Palphabeta could be stoichiometrically phosphorylated in vitro. Pgamma phosphorylation has modest effects on its binding affinity to Palphabeta and its overall inhibitory potency. However, phosphorylation at Thr22 mildly decreases the ability of the central region of Pgamma to bind to Palphabeta and to stabilize cGMP binding at the GAF domains. Similar to PDE5, phosphorylation of photoreceptor Palphabeta regulates cGMP binding at the GAF domains. These results suggest a potential role of Palphabeta phosphorylation in regulating PDE by regulating cGMP levels in photoreceptor cells

Asymptotic enumeration of some RNA secondary structures

AbstractIn this paper, we derive recursions of some RNA secondary structures with certain properties under two new representations. Furthermore, by making use of methods of asymptotic analysis and generating functions we present asymptotic enumeration of these RNA secondary structures

Dedifferentiation of committed epithelial cells into stem cells in vivo

Summary Cellular plasticity contributes to the regenerative capacity of plants, invertebrates, teleost fishes, and amphibians. In vertebrates, differentiated cells are known to revert into replicating progenitors, but these cells do not persist as stable stem cells. We now present evidence that differentiated airway epithelial cells can revert into stable and functional stem cells in vivo. Following the ablation of airway stem cells, we observed a surprising increase in the proliferation of committed secretory cells. Subsequent lineage tracing demonstrated that the luminal secretory cells had dedifferentiated into basal stem cells. Dedifferentiated cells were morphologically indistinguishable from stem cells and they functioned as well as their endogenous counterparts to repair epithelial injury. Indeed, single secretory cells clonally dedifferentiated into multipotent stem cells when they were cultured ex vivo without basal stem cells. In contrast, direct contact with a single basal stem cell was sufficient to prevent secretory cell dedifferentiation. In analogy to classical descriptions of amphibian nuclear reprogramming, the propensity of committed cells to dedifferentiate was inversely correlated to their state of maturity. This capacity of committed cells to dedifferentiate into stem cells may play a more general role in the regeneration of many tissues and in multiple disease states, notably cancer

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases

Development of a Primary Human Co-Culture Model of Inflamed Airway Mucosa

Neutrophil breach of the mucosal surface is a common pathological consequence of infection. We present an advanced co-culture model to explore neutrophil transepithelial migration utilizing airway mucosal barriers differentiated from primary human airway basal cells and examined by advanced imaging. Human airway basal cells were differentiated and cultured at air-liquid interface (ALI) on the underside of 3 µm pore-sized transwells, compatible with the study of transmigrating neutrophils. Inverted ALIs exhibit beating cilia and mucus production, consistent with conventional ALIs, as visualized by micro-optical coherence tomography (µOCT). µOCT is a recently developed imaging modality with the capacity for real time two- and three-dimensional analysis of cellular events in marked detail, including neutrophil transmigratory dynamics. Further, the newly devised and imaged primary co-culture model recapitulates key molecular mechanisms that underlie bacteria-induced neutrophil transepithelial migration previously characterized using cell line-based models. Neutrophils respond to imposed chemotactic gradients, and migrate in response to Pseudomonas aeruginosa infection of primary ALI barriers through a hepoxilin A3-directed mechanism. This primary cell-based co-culture system combined with µOCT imaging offers significant opportunity to probe, in great detail, micro-anatomical and mechanistic features of bacteria-induced neutrophil transepithelial migration and other important immunological and physiological processes at the mucosal surface

The catalytic and GAF domains of the rod cGMP phosphodiesterase (PDE6) heterodimer are regulated by distinct regions of its inhibitory gamma subunit

The central effector of visual transduction in retinal rod photoreceptors, cGMP phosphodiesterase (PDE6), is a catalytic heterodimer (alpha beta) to which low molecular weight inhibitory gamma subunits bind to form the nonactivated PDE holoenzyme (alpha beta gamma (2)). Although it is known that gamma binds tightly to alpha beta, the binding affinity for each gamma subunit to alpha beta, the domains on gamma that interact with alpha beta, and the allosteric interactions between gamma and the regulatory and catalytic regions on alpha beta are not well understood. We show here that the gamma subunit binds to two distinct sites on the catalytic alpha beta dimer (K-D1 to alpha beta is absent when cAMP occupies the noncatalytic sites. Two major domains on gamma can interact independently with alpha beta with the N-terminal half of gamma binding with 50-fold greater affinity than its C-terminal, inhibitory region. The N-terminal half of gamma is responsible for the positive cooperativity between gamma and cGMP binding sites on alpha beta but has no effect on catalytic activity. Using synthetic peptides, we identified regions of the amino acid sequence of gamma that bind to alpha beta, restore high affinity cGMP binding to low affinity noncatalytic sites, and retard cGMP exchange with both noncatalytic sites. Subunit heterogeneity, multiple sites of gamma interaction with alpha beta, and positive cooperativity of gamma with the GAF domains are all likely to contribute to precisely controlling the activation and inactivation kinetics of PDE6 during visual transduction in rod photoreceptors

Gbdmr: identifying differentially methylated CpG regions in the human genome via generalized beta regressions

Abstract Background DNA methylation is a biochemical process in which a methyl group is added to the cytosine-phosphate-guanine (CpG) site on DNA molecules without altering the DNA sequence. Multiple CpG sites in a certain genome region can be differentially methylated across phenotypes. Identifying these differentially methylated CpG regions (DMRs) associated with the phenotypes contributes to disease prediction and precision medicine development. Results We propose a novel DMR detection algorithm, gbdmr. In contrast to existing methods under a linear regression framework, gbdmr assumes that DNA methylation levels follow a generalized beta distribution. We compare gbdmr to alternative approaches via simulations and real data analyses, including dmrff, a new DMR detection approach that shows promising performance among competitors, and the traditional EWAS that focuses on single CpG sites. Our simulations demonstrate that gbdmr is superior to the other two when the correlation between neighboring CpG sites is strong, while dmrff shows a higher power when the correlation is weak. We provide an explanation of these phenomena from a theoretical perspective. We further applied the three methods to multiple real DNA methylation datasets. One is from a birth cohort study undertaken on the Isle of Wight, United Kingdom, and the other two are from the Gene Expression Omnibus database repository. Overall, gbdmr identifies more DMR CpGs linked to phenotypes than dmrff, and the simulated results support the findings. Conclusions Gbdmr is an innovative method for detecting DMRs based on generalized beta regression. It demonstrated notable advantages over dmrff and traditional EWAS, particularly when adjacent CpGs exhibited moderate to strong correlations. Our real data analyses and simulated findings highlight the reliability of gbdmr as a robust DMR detection tool. The gbdmr approach is accessible and implemented by R on GitHub: https://github.com/chengzhouwu/gbdmr

IDENTIFYING INTERGENERATIONAL PATTERNS OF CORRELATED METHYLATION SITES

DNA methylation can be transmitted through generations. This paper proposes a clustering method to identify the intergenerational patterns from parents to their offspring. Motivated by the potential of correlation between DNA methylation sites, we use the multivariate generalized beta distribution to model the blockwise correlation structure among the sites. A stochastic EM algorithm is implemented to estimate the parameters, and BIC is applied to determine the optimal number of clusters. Simulations demonstrate the feasibility of the proposed method. We further applied the approach to cluster DNA methylation data generated from a cohort study on asthma and allergic conditions

Efficacy of Alfalfa Saponins on Promoting Pigmentation by Astaxanthin in Blood Parrot Fish (Vieja synspila♀× Amphilophus citrinellus♂)

This study was conducted to evaluate the effect of alfalfa saponins on pigmentation induced by astaxanthin in 540 blood parrot fish (Vieja synspila ♀ × Amphilophus citrinellus ♂). The fish were divided into six treatment groups: basal diet; a diet supplemented with 3‰ astaxanthin; three diets with 3‰ astaxanthin plus 3‰, 6‰ or 12‰ alfalfa saponins respectively; and a diet supplemented with 4‰ alfalfa saponins alone. The fish were maintained in three aquaria per treatment. At 20, 40, 60, and 80 days, 6 blood parrot fish per aquarium were sampled. At the end of the experiment, the carotenoid content of scales, skin, and caudal fin in groups supplied with 3‰ astaxanthin plus alfalfa saponins were significantly higher than the control. The redness values, a*, of the body and caudal fins in these groups increased significantly. Conversely, the cholesterol and triglyceride content decreased significantly. Non-esterified fatty acid content and lysozyme activity were significantly higher in the three groups fed supplemented astaxanthin diets with the addition of alfalfa saponins, than the group fed a diet supplemented with 3‰ astaxanthin alone (P<0.05). The results show that the addition of alfalfa saponins to diets supplemented with astaxanthin, improves astaxanthin absorption and utilization

Regulation of photoreceptor phosphodiesterase (PDE6) by phosphorylation of its inhibitory gamma subunit re-evaluated

Phosphorylation of the inhibitory gamma subunit (Pgamma) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pgamma phosphorylation for PDE6 regulation by preparing Pgamma stoichiometrically phosphorylated at Thr(22) or at Thr(35). Phosphorylation of Pgamma at either residue caused a minor decrease-not the previously reported increase-in the ability of Pgamma to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pgamma phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pgamma subunits. The strength of Pgamma interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pgamma phosphorylation at Thr(22) (but not Thr(35)), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527-27534). In contrast, the effects of Pgamma phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pgamma at either Thr(22) or Thr(35) greatly diminished its ability to bind activated transducin, consistent with earlier work. In situ phosphorylation of Pgamma by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only similar to10% of the endogenous Pgamma was phosphorylated. This is attributed to Pgamma being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pgamma phosphorylation at either Thr(22) or Thr(35) modestly weakens its direct interactions with PDE6. However, Pgamma phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pgamma available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802-37808) in photoreceptor cells