Effects of prenatal Poly I:C exposure on global histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity in the mouse brain

The aim of our study was to investigate the brain-specific epigenetic effects on global enzymatic histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity after prenatal exposure to maternal immune challenge by polyinosinic:polycytidylic acid (Poly I:C) at gestational day (GD) 17 in C57BL/6JRccHsd mouse offspring. Pregnant mice were randomly divided into 2 groups, receiving either 5 mg/kg Poly I:C or phosphate buffered saline (PBS) intravenously at GD 17. Subsequently, the effects on whole brain enzymatic HDAC and DNMT activity and the protein levels of various HDAC isoforms were assessed in the offspring. Overall, a significant sex × treatment interaction effect was observed after prenatal exposure to maternal immune challenge by Poly I:C, indicative of increased global HDAC activity particularly in female offspring from mothers injected with Poly I:C when compared to controls. Results on the levels of specific HDAC isoforms suggested that neither differences in the levels of HDAC1, HDAC2, HDAC3, HDAC4 or HDAC6 could explain the increased global HDAC activity observed in female Poly I:C offspring. In conclusion, we show that Poly I:C administration to pregnant mice alters global brain HDAC, but not DNMT activity in adult offspring, whereas it is still unclear which specific HDAC(s) mediate(s) this effect. These results indicate the necessity for further research on the epigenetic effects of Poly I:C.     

Characterization of reticulofibroblastoid colonies (CFU-RF) derived from bone marrow and long-term marrow culture monolayers

The maintenance of hemopoietic precursors in long-term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose-clotted plasma in the presence of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU-RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid-laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU-F). The relationship of CFU-RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU-RF-derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU-RF-derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU-RF is not part of the repertoire of hemopoietic stem cells.     

Assessment of the hybridization between rhesus (Macaca mulatta) and long‐tailed macaques (M. fascicularis) based on morphological characters

The evolution of human skin pigmentation must address both the initial evolution of intense epidermal pigmentation in hominins, and its subsequent dilution in modern humans. While many authorities believe that epidermal pigmentation evolved to protect against either ultraviolet B (UV‐B) irradiation‐induced mutagenesis or folic acid photolysis, we hypothesize that pigmentation augmented the epidermal barriers by shifting the UV‐B dose–response curve from toxic to beneficial. Whereas erythemogenic UV‐B doses produce apoptosis and cell death, suberythemogenic doses benefit permeability and antimicrobial function. Heavily melanized melanocytes acidify the outer epidermis and emit paracrine signals that augment barrier competence. Modern humans, residing in the cooler, wetter climes of south‐central Europe and Asia, initially retained substantial pigmentation. While their outdoor lifestyles still permitted sufficient cutaneous vitamin D3 (VD3) synthesis, their marginal nutritional status, coupled with cold‐induced caloric needs, selected for moderate pigment reductions that diverted limited nutritional resources towards more urgent priorities (=metabolic conservation). The further pigment‐dilution that evolved as humans reached north‐central Europe (i.e., northern France, Germany), likely facilitated cutaneous VD3 synthesis, while also supporting ongoing, nutritional requirements. But at still higher European latitudes where little UV‐B breaches the atmosphere (i.e., present‐day UK, Scandinavia, Baltic States), pigment dilution alone could not suffice. There, other nonpigment‐related mutations evolved to facilitate VD3 production; for example, in the epidermal protein, filaggrin, resulting in reduced levels of its distal metabolite, trans‐urocanic acid, a potent UV‐B chromophore. Thus, changes in human pigmentation reflect a complex interplay between latitude, climate, diet, lifestyle, and shifting metabolic priorities.     

Selectivity in Enrichment of cAMP-dependent Protein Kinase Regulatory Subunits Type I and Type II and Their Interactors Using Modified cAMP Affinity Resins

cAMP regulates cellular functions primarily by activating PKA. The involvement of PKAs in various signaling pathways occurring simultaneously in different cellular compartments necessitates stringent spatial and temporal regulation. This specificity is largely achieved by binding of PKA to protein scaffolds, whereby a distinct group of proteins called A kinase anchoring proteins (AKAPs) play a dominant role. AKAPs are a diverse family of proteins that all bind via a small PKA binding domain to the regulatory subunits of PKA. The binding affinities between PKA and several AKAPs can be different for different isoforms of the regulatory subunits of PKA. Here we employ a combination of affinity chromatography and mass spectrometry-based quantitative proteomics to investigate specificity in PKA-AKAP interactions. Three different immobilized cAMP analogs were used to enrich for PKA and its interacting proteins from several systems; HEK293 and RCC10 cells and rat lung and testis tissues. Stable isotope labeling was used to confidently identify and differentially quantify target proteins and their preferential binding affinity for the three different cAMP analogs. We were able to enrich all four isoforms of the regulatory subunits of PKA and concomitantly identify more than 10 AKAPs. A selective enrichment of the PKA RI isoforms could be achieved; which allowed us to unravel which AKAPs bind preferentially to the RI or RII regulatory domains of PKA. Of the twelve AKAPs detected, seven preferentially bound to RII, whereas the remaining five displayed at least dual specificity with a potential preference for RI. For some of these AKAPs our data provide the first insights into their specificity.cAMP is an ubiquitous second messenger that transduces signals from a variety of hormones, neurotransmitters, and inflammatory mediators to regulate a large number of key cellular processes. cAMP can influence cell growth, differentiation, and movement as well as regulating specialized actions unique to specific cell types. The principal target of cAMP is cAMP-dependent protein kinase (PKA)¹. Several other proteins such as cyclic nucleotide gated ion channels (1), phosphodiesterases (PDE) (2), and guanine nucleotide exchange factors (Epac) (3) bind cAMP. Interestingly, localized pools of cAMP regulate defined physiological events. It appears that for such events a supramolecular complex is required that comprises of the appropriate effector system together with signal termination enzymes such as PDEs and phosphatases that are sequestered by scaffolding proteins (4). Some of the best described scaffolding proteins are the so-called A-kinase anchoring proteins (AKAPs), which all bind specifically to the N-terminal dimerization domain of the PKA regulatory domain. The organization of a few of these individual supramolecular complexes containing PKA/AKAPs/PDE etc. has been described (4); numerous more of such complexes are expected to exist.The regulatory domains of mammalian PKAs exist in several isoforms such as RIα, RIβ, RIIα, and RIIβ, which are all encoded by separate genes. The two major isoforms i.e. RI and RII differ in molecular weight, isoelectric point, amino acid sequence, phosphorylation status, tissue distribution, and sub-cellular localization. RI and RII subunits are known to bind to AKAPs with distinct levels of affinity adding another level of intracellular organization for PKA and also facilitating the diversity of the cAMP-mediated signal transduction pathways. Although the PKA-R isoforms differ in functionality, they share a similar overall organization i.e. a dimerization domain, the catalytic subunits inhibitor region, and two cAMP binding domains. The two cAMP binding domains differ in cAMP binding kinetics and are known as site A and site B, respectively (5). Both sites share considerable sequence identity, as a result of a tandem gene duplication, and have conserved phosphate binding cassettes that can be considered as signature motif for cAMP binding. The relative orientation of these two sites is nonetheless, quite different in RI and RII. Additionally, A and B sites have different binding affinity to cAMP derivatives. Site A has a preference for N6-substituted analogs whereas site B is preferred by C2- and C8-substituted analogs (6).PKA has been studied extensively (7, 8). One of the important goals therein is to develop different cAMP analogs that can result in specific binding, activation, and/or inhibition for each individual cAMP interaction site of the RI and RII isoforms (9). This can help to decipher in detail specific cyclic nucleotide signaling pathways (10). To fully interpret such pathways, analogs should ideally not cross-activate (or inhibit) with other cAMP-regulated proteins such as the before mentioned PDEs, Epac, cyclic nucleotide gated ion channels, and the cGMP-dependent protein kinase (PKG). Although the latter is mainly activated by cGMP, it also binds to cAMP (11, 12). It has been suggested that cGMP and cAMP can cross-activate their respective kinases (13). This cross-talk between PKA and PKG hampers, to some degree, the study of these proteins individually, as dissecting the individual pathways of PKA and PKG requires specific binders, activators, or inhibitors (9, 14). Compared with PKA, PKG is involved in quite different signaling pathways, such as the well characterized nitric oxide-mediated relaxation of smooth muscle cells (15).The development of synthetic cAMP and cGMP analogs as tools to unravel specific signal transduction pathways requires the sensitive identification and characterization of their cyclic nucleotide interacting proteins. These proteins are typically relatively low abundant, and therefore specific enrichment techniques are essential to study these so-called cyclic-nucleotide interactomes. In recent years such affinity enrichment techniques have been coupled to sensitive mass spectrometric identification of the enriched proteins, nowadays often referred to as chemical proteomics. Chemical proteomics using small molecules as baits, i.e. messenger molecules, drugs, or metabolites, becomes more and more widely used to selectively isolate target proteins from whole cell lysates enabling the analysis of protein subcomplexes and/or signaling pathways (16, 17).In the present study we compare the properties of three cAMP analogs immobilized individually on agarose beads, for enrichment, isolation, and detection of cyclic nucleotide interacting proteins and their interaction partners, like AKAPs, directly from a crude lysate of cells and tissue. To quantify differential affinity, we use a common strategy in proteomics, namely stable isotope labeling, whereby we introduce the label via reductive amination (18–20). Most interestingly, a very selective enrichment of PKA RI isoforms can be achieved by using cAMP-agarose beads in which the hydroxyl group at the 2′ position on the ribose was replaced with a methoxyl group. This allows us to distinguish, which AKAPs bind preferentially to the RI or RII isoforms. Therefore, this approach provides an elegant tool to further decipher specific cyclic nucleotide signaling pathways.