ATP interacts with the CPVT mutation-associated central domain of the cardiac ryanodine receptor

Background

This study was designed to determine whether the cardiac ryanodine receptor (RyR2) central domain, a region associated with catecholamine polymorphic ventricular tachycardia (CPVT) mutations, interacts with the RyR2 regulators, ATP and the FK506-binding protein 12.6 (FKBP12.6).

Methods

Wild-type (WT) RyR2 central domain constructs (G²²³⁶to G²⁴⁹¹) and those containing the CPVT mutations P2328S and N2386I, were expressed as recombinant proteins. Folding and stability of the proteins were examined by circular dichroism (CD) spectroscopy and guanidine hydrochloride chemical denaturation.

Results

The far-UV CD spectra showed a soluble stably-folded protein with WT and mutant proteins exhibiting a similar secondary structure. Chemical denaturation analysis also confirmed a stable protein for both WT and mutant constructs with similar two-state unfolding. ATP and caffeine binding was measured by fluorescence spectroscopy. Both ATP and caffeine bound with an EC₅₀ of ~ 200–400 μM, and the affinity was the same for WT and mutant constructs. Sequence alignment with other ATP binding proteins indicated the RyR2 central domain contains the signature of an ATP binding pocket. Interaction of the central domain with FKBP12.6 was tested by glutaraldehyde cross-linking and no association was found.

Conclusions

The RyR2 central domain, expressed as a ‘correctly’ folded recombinant protein, bound ATP in accord with bioinformatics evidence of conserved ATP binding sequence motifs. An interaction with FKBP12.6 was not evident. CPVT mutations did not disrupt the secondary structure nor binding to ATP.

General significance

Part of the RyR2 central domain CPVT mutation cluster, can be expressed independently with retention of ATP binding.     

Generalist insects behave in a jasmonate-dependent manner on their host plants,leaving induced areas quickly and staying longer on distant parts

Plants are sessile, so have evolved sensitive ways to detect attacking herbivores and sophisticated strategies to effectively defend themselves. Insect herbivory induces synthesis of the phytohormone jasmonic acid which activates downstream metabolic pathways for various chemical defences such as toxins and digestion inhibitors. Insects are also sophisticated animals, and many have coevolved physiological adaptations that negate this induced plant defence. Insect behaviour has rarely been studied in the context of induced plant defence, although behavioural adaptation to induced plant chemistry may allow insects to bypass the host''s defence system. By visualizing jasmonate-responsive gene expression within whole plants, we uncovered spatial and temporal limits to the systemic spread of plant chemical defence following herbivory. By carefully tracking insect movement, we found induced changes in plant chemistry were detected by generalist Helicoverpa armigera insects which then modified their behaviour in response, moving away from induced parts and staying longer on uninduced parts of the same plant. This study reveals that there are plant-wide signals rapidly generated following herbivory that allow insects to detect the heterogeneity of plant chemical defences. Some insects use these signals to move around the plant, avoiding localized sites of induction and staying ahead of induced toxic metabolites.     

Environment-dependent intralocus sexual conflict in a dioecious plant

Intralocus sexual conflict is a form of conflict that does not involve direct interactions between males and females. It arises when selection on a shared trait with a common genetic basis differs between the sexes. Environmental factors, such as resource availability, may influence the expression and evolutionary outcome of such conflict. We quantified the genetic variance-covariance matrix, G, for both sexes of Silene latifolia for floral and leaf traits, as well as the between-sex matrix, B. We also quantified selection on the sexes via survival for 2 yr in four natural populations that varied in water availability. Environment-dependent intralocus sexual conflict exists for specific leaf area, a trait that is genetically correlated between the sexes. Males experienced significant negative selection, but only in populations with relatively limited water availability. Females experienced weakly positive or significant stabilizing selection on the same trait. Specific leaf area is genetically correlated with flower size and number, which are sexually dimorphic in this species. The extent of intralocus sexual conflict varied with the environment. Resolution of such conflict is likely to be confounded, given that specific leaf area is highly genetically integrated with other traits that are also divergent between the sexes.     

β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway

The role of the β2 adrenergic receptor (β2AR) in the regulation of chronic neurodegenerative inflammation within the CNS is poorly understood. The purpose of this study was to determine neuroprotective effects of long-acting β2AR agonists such as salmeterol in rodent models of Parkinson's disease. Results showed salmeterol exerted potent neuroprotection against both LPS and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/1-methyl-4-phenylpyridinium-induced dopaminergic neurotoxicity both in primary neuron-glia cultures (at subnanomolar concentrations) and in mice (1-10 μg/kg/day doses). Further studies demonstrated that salmeterol-mediated neuroprotection is not a direct effect on neurons; instead, it is mediated through the inhibition of LPS-induced microglial activation. Salmeterol significantly inhibited LPS-induced production of microglial proinflammatory neurotoxic mediators, such as TNF-α, superoxide, and NO, as well as the inhibition of TAK1-mediated phosphorylation of MAPK and p65 NF-κB. The anti-inflammatory effects of salmeterol required β2AR expression in microglia but were not mediated through the conventional G protein-coupled receptor/cAMP pathway. Rather, salmeterol failed to induce microglial cAMP production, could not be reversed by either protein kinase A inhibitors or an exchange protein directly activated by cAMP agonist, and was dependent on β-arrestin2 expression. Taken together, our results demonstrate that administration of extremely low doses of salmeterol exhibit potent neuroprotective effects by inhibiting microglial cell activation through a β2AR/β-arrestin2-dependent but cAMP/protein kinase A-independent pathway.     

Suppressor of cytokine signaling 1 regulates embryonic myelopoiesis independently of its effects on T cell development

Suppressor of cytokine signaling 1 (SOCS1) has been shown to play important roles in the immune system. It acts as a key negative regulator of signaling via receptors for IFNs and other cytokines controlling T cell development, as well as Toll receptor signaling in macrophages and other immune cells. To gain further insight into SOCS1, we have identified and characterized the zebrafish socs1 gene, which exhibited sequence and functional conservation with its mammalian counterparts. Initially maternally derived, the socs1 gene showed early zygotic expression in mesodermal structures, including the posterior intermediate cell mass, a site of primitive hematopoiesis. At later time points, expression was seen in a broad anterior domain, liver, notochord, and intersegmental vesicles. Morpholino-mediated knockdown of socs1 resulted in perturbation of specific hematopoietic populations prior to the commencement of lymphopoiesis, ruling out T cell involvement. However, socs1 knockdown also lead to a reduction in the size of the developing thymus later in embryogenesis. Zebrafish SOCS1 was shown to be able to interact with both zebrafish Jak2a and Stat5.1 in vitro and in vivo. These studies demonstrate a conserved role for SOCS1 in T cell development and suggest a novel T cell-independent function in embryonic myelopoiesis mediated, at least in part, via its effects on receptors using the Jak2-Stat5 pathway.     

The influence of bisnaphthalimidopropyl polyamines on DNA instability and repair in Caco-2 colon epithelial cells

Bisnaphthalimido compounds bis-intercalate to DNA via the major groove and are potentially potent cancer therapeutics. Previously, we incorporated natural polyamines as linkers connecting the two naphthalimido ring moieties to create a series of soluble bisnaphthalimidopropyl polyamines (BNIPPs). Here, extending earlier work on bisnaphthalimidopropylspermidine (BNIPSpd)-induced apoptosis in colon adenocarcinoma Caco-2 cells, we compare the cytotoxicity and genotoxicity of BNIPSpd relative to the spermine and oxaspermine derivatives, bisnaphthalimidopropylspermine (BNIPSpm) and bisnaphthalimidopropyloxaspermine (BNIPOSpm). The order of cytotoxicity after 24 h was BNIPSpd (IC₅₀ = 0.47 μM) > BNIPSpm (IC₅₀ = 10.04 μM) > BNIPOSpm (IC₅₀ >50 μM). After a 72-h BNIPOSpm exposure, an IC₅₀ = 10.25 μM was achieved. With 4-h exposure to BNIPSpd or BNIPSpm or 12-h exposure to BNIPOSpm, concentrations ≥1 μM induced a significant dose-dependent increase in DNA damage as measured by alkaline single-cell gel electrophoresis. The longer incubation times required for BNIPOSpm to induce DNA strand breaks reflect a slower rate of BNIPOSpm cellular distribution as monitored via BNIPP fluorescence within the cells. Moreover, exposure to a non-genotoxic concentration of BNIPSpd, BNIPSpm (0.1 μM for 4 h) or BNIPOSpm (0.1 μM for 12 h) induced a significant decrease in repair of oxidative DNA damage induced by hydrogen peroxide. In conclusion, BNIPP exposure in Caco-2 cells is associated with significant induction of DNA damage and inhibition of DNA repair at non-genotoxic concentrations. The latter is a novel consequence of BNIPP–cell interactions which adds to the spectrum of therapeutically relevant activities that may be exploited for the design and development of naphthalimide-based therapeutics.