Roles of surface residues of intracellular domains of heag potassium channels

Ether-a-go-go potassium channels have large intracellular regions containing ‘Per-Ant-Sim’ (PAS) and cyclic nucleotide binding (cNBD) domains at the N- and C-termini, respectively. In heag1 and heag2 channels, recent studies have suggested that the N- and C-terminal domains interact, and affect activation properties. Here, we have studied the effect of mutations of residues on the surfaces of PAS and cNBD domains. For this, we introduced alanine and lysine mutations in heag1 channels, and recorded currents by two-electrode voltage clamp. In both the PAS domain and the cNBD domain, contiguous areas of conserved residues on the surfaces of these domains were found which affected the activation kinetics of the channel. Next, we investigated possible effects of mutations on domain interactions of PAS and cNBD proteins in heag2 by co-expressing these domain proteins followed by analysis with native gels and western blotting. We found oligomeric association between these domains. Mutations F30A and A609K (on the surfaces of the PAS and cNBD domains, respectively) affected oligomeric compositions of these domains when proteins for PAS and cNBD domains were expressed together. Taken together, the data suggest that the PAS and cNBD domains form interacting oligomers that have roles in channel function.     

Lysophosphatidic acid-induced arterial wall remodeling: Requirement of PPARγ but not LPA1 or LPA2 GPCR

Lysophosphatidic acid (LPA) and its ether analog alkyl-glycerophosphate (AGP) elicit arterial wall remodeling when applied intralumenally into the uninjured carotid artery. LPA is the ligand of eight GPCRs and the peroxisome proliferator-activated receptor γ (PPARγ). We pursued a gene knockout strategy to identify the LPA receptor subtypes necessary for the neointimal response in a non-injury model of carotid remodeling and also compared the effects of AGP and the PPARγ agonist rosiglitazone (ROSI) on balloon injury-elicited neointima development. In the balloon injury model AGP significantly increased neointima; however, rosiglitazone application attenuated it. AGP and ROSI were also applied intralumenally for 1 h without injury into the carotid arteries of LPA₁, LPA₂, LPA_1&2 double knockout, and Mx1Cre-inducible conditional PPARγ knockout mice targeted to vascular smooth muscle cells, macrophages, and endothelial cells. The neointima was quantified and also stained for CD31, CD68, CD11b, and α-smooth muscle actin markers. In LPA₁, LPA₂, LPA_1&2 GPCR knockout, Mx1Cre transgenic, PPARγ^fl/−, and uninduced Mx1Cre × PPARγ^fl/− mice AGP- and ROSI-elicited neointima was indistinguishable in its progression and cytological features from that of WT C57BL/6 mice. In PPARγ^−/− knockout mice, generated by activation of Mx1Cre-mediated recombination, AGP and ROSI failed to elicit neointima and vascular wall remodeling. Our findings point to a difference in the effects of AGP and ROSI between the balloon injury- and the non-injury chemically-induced neointima. The present data provide genetic evidence for the requirement of PPARγ in AGP- and ROSI-elicited neointimal thickening in the non-injury model and reveal that the overwhelming majority of the cells in the neointimal layer express α-smooth muscle actin.     

Evidence of Symbiosis Between the Soil Yeast <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> and a Sclerophyllous Medicinal Shrub, <Emphasis Type="Italic">Agathosma betulina</Emphasis> (Berg.) Pillans

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.Cyclic GMP (cGMP)-dependent protein kinases (PKGs) are the major intracellular mediators of cGMP signal transduction in eukaryotic cells. Mammalian PKGs regulate a number of physiological processes including smooth muscle relaxation, platelet aggregation, intestinal secretion and hippocampal and cerebellar learning (reviewed in reference 27). Unlike cAMP-dependent protein kinase (PKA), PKG comprises both a catalytic domain and a regulatory domain in a single polypeptide. In the inactivated state, part of the regulatory domain (a substrate-like sequence known as the autoinhibitory domain) is thought to be bound to the catalytic domain. This binding is released and the enzyme activated upon binding of cGMP to the regulatory domain and opening of the substrate-binding region within the catalytic domain (1, 14). The PKG of the malaria parasite Plasmodium falciparum (PfPKG) and orthologues from the related coccidian parasites Toxoplasma and Eimeria are encoded by a single-copy gene and have been shown to differ from the mammalian enzymes in several respects. These include a larger number of cGMP binding sites (three functional plus one degenerate) in the regulatory domain of the apicomplexan enzymes (6, 7, 17), highly cooperative stimulation by cGMP (8, 17), and relative insensitivity to 8-substituted cGMP analogues (6, 7, 26). The parasite isoforms also lack an N-terminal leucine zipper motif that mediates homodimerization in mammalian isoforms, and evidence suggests that they are monomeric (17). Interestingly, it has been shown that the coccidian PKGs exist as both cytosolic and membrane-associated (mediated by N-terminal myristoylation and palmitoylation) isoforms (12), but the amino acid motifs required for these modifications are absent in the PfPKG sequence, and it seems to lack acylated forms (8).The coccidian PKGs have been shown to be the target of a potent anticoccidial agent, 4-[2-(4-fluorphenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl]pyridine (compound 1), which is a competitive inhibitor of ATP binding (17). The selectivity of the compound for apicomplexan enzymes is attributed to the relative accessibility of a hydrophobic pocket that overlaps with the ATP binding site conferred by a critical threonine residue in the key “gatekeeper” position (T761 in Toxoplasma gondii; T770 in Eimeria tenella) (11). Access is thought to be prevented by the relatively bulky side chain of the residue that occupies the corresponding position in mammalian PKGs. Expression of compound 1-insensitive coccidian PKGs (harboring T/Q or T/M gatekeeper residue substitutions) in Toxoplasma abolished the effects of the inhibitor on the parasite, thus establishing that PKG is the primary target of compound 1 in T. gondii. These compound 1-insensitive mutants were used to determine a role for coccidian PKG in secretion of micronemal adhesive proteins, attachment to and invasion of host cells and gliding motility of E. tenella sporozoites and T. gondii tachyzoites. Compound 1 has also been shown to be a potent inhibitor of the native P. falciparum PKG enzyme (50% inhibitory concentration [IC₅₀] of 8.53 nM on cGMP-dependent kinase activity in purified fractions using a fixed ATP concentration of 5 μM in the assay) but has limited in vivo activity in the rodent malaria model P. berghei (8).P. falciparum causes the most serious form of malaria in humans and is responsible for the deaths of approximately one million people each year. The parasite life cycle is complex consisting of distinct phases in the mosquito vector and the human host. Pathogenesis is caused by asexual parasites which proliferate within red blood cells. The invasive form (merozoite) enters a red blood cell and forms a ring-stage parasite. This develops into a trophozoite which feeds on hemoglobin, and the resulting schizont releases up to 32 daughter merozoites in a 48-h cycle. A small proportion of the schizonts release merozoites that differentiate into male or female gamete precursors (gametocytes). These sexual cells are essential for malaria transmission and must be taken up by an Anopheles mosquito to continue the life cycle. Upon entering the insect midgut, environmental cues trigger gametogenesis. Recently, we reported that PfPKG plays an essential role in initiating this process. The initial morphological change after activation of P. falciparum gametocytes, from crescent-shaped to spherical (known as rounding up), is inhibited by compound 1. Genetically manipulated parasites expressing a mutant (T618Q gatekeeper substitution) compound 1-insensitive but fully active PKG, introduced by allelic replacement, were resistant to the effects of compound 1 on gametogenesis, demonstrating a direct role for the enzyme in the initiation of this process (23).There is also evidence suggesting a role for PKG in P. falciparum erythrocytic stages. It has been reported that PfPKG is expressed at both the mRNA and the protein level in asexual blood stages, as well as the sexual phase of the life cycle (6, 8). Although the PfPKG gene could be targeted for allelic replacement, we were unable to disrupt it, suggesting an essential role for PKG in P. falciparum asexual blood stage development since it is this life cycle stage on which transfection and drug selection of genetically modified parasites are performed. Furthermore, we and others (8, 23) have observed that compound 1 inhibits in vitro-cultured P. falciparum (IC₅₀s in the sub-low-μM range depending on the isolate). Although the role of PKG in the initiation of gametogenesis has been elucidated, its role in erythrocytic stages was unknown prior to the present study.We demonstrate here that PfPKG has a central role in the late stages of erythrocytic schizogony. Compound 1-treated P. falciparum parasites develop normally through the trophozoite stage but arrest late in schizont development, forming large dysmorphic schizonts. Genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG are resistant to the effects of both compound 1 and (a second imidazopyridine PKG inhibitor) compound 2 on schizogony but not to the broad-specificity serine/threonine kinase inhibitor staurosporine. This demonstrates that PfPKG is the primary target of the inhibitors and provides direct evidence that this enzyme is essential for progression of the asexual erythrocytic stage of the malaria parasite life cycle.     

Structure-guided design of α-amino acid-derived Pin1 inhibitors

The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics. Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of α-benzimidazolyl-substituted amino acids. Examples inhibited Pin1 activity with IC₅₀ <100 nM, but were inactive on cells. Replacement of the benzimidazole ring with a naphthyl group resulted in a 10–50-fold loss in ligand potency, but these examples downregulated biomarkers of Pin1 activity and blocked proliferation of PC3 cells.     

Rapid Changes in the Light/Dark Cycle Disrupt Memory of Conditioned Fear in Mice

Background

Circadian rhythms govern many aspects of physiology and behavior including cognitive processes. Components of neural circuits involved in learning and memory, e.g., the amygdala and the hippocampus, exhibit circadian rhythms in gene expression and signaling pathways. The functional significance of these rhythms is still not understood. In the present study, we sought to determine the impact of transiently disrupting the circadian system by shifting the light/dark (LD) cycle. Such “jet lag” treatments alter daily rhythms of gene expression that underlie circadian oscillations as well as disrupt the synchrony between the multiple oscillators found within the body.

Methodology/Principal Findings

We subjected adult male C57Bl/6 mice to a contextual fear conditioning protocol either before or after acute phase shifts of the LD cycle. As part of this study, we examined the impact of phase advances and phase delays, and the effects of different magnitudes of phase shifts. Under all conditions tested, we found that recall of fear conditioned behavior was specifically affected by the jet lag. We found that phase shifts potentiated the stress-evoked corticosterone response without altering baseline levels of this hormone. The jet lag treatment did not result in overall sleep deprivation, but altered the temporal distribution of sleep. Finally, we found that prior experience of jet lag helps to compensate for the reduced recall due to acute phase shifts.

Conclusions/Significance

Acute changes to the LD cycle affect the recall of fear-conditioned behavior. This suggests that a synchronized circadian system may be broadly important for normal cognition and that the consolidation of memories may be particularly sensitive to disruptions of circadian timing.     

Toward a global view of alcohol, tobacco, cannabis, and cocaine use: findings from the WHO World Mental Health Surveys

Background

Alcohol, tobacco, and illegal drug use cause considerable morbidity and mortality, but good cross-national epidemiological data are limited. This paper describes such data from the first 17 countries participating in the World Health Organization''s (WHO''s) World Mental Health (WMH) Survey Initiative.

Methods and Findings

Household surveys with a combined sample size of 85,052 were carried out in the Americas (Colombia, Mexico, United States), Europe (Belgium, France, Germany, Italy, Netherlands, Spain, Ukraine), Middle East and Africa (Israel, Lebanon, Nigeria, South Africa), Asia (Japan, People''s Republic of China), and Oceania (New Zealand). The WHO Composite International Diagnostic Interview (CIDI) was used to assess the prevalence and correlates of a wide variety of mental and substance disorders. This paper focuses on lifetime use and age of initiation of tobacco, alcohol, cannabis, and cocaine. Alcohol had been used by most in the Americas, Europe, Japan, and New Zealand, with smaller proportions in the Middle East, Africa, and China. Cannabis use in the US and New Zealand (both 42%) was far higher than in any other country. The US was also an outlier in cocaine use (16%). Males were more likely than females to have used drugs; and a sex–cohort interaction was observed, whereby not only were younger cohorts more likely to use all drugs, but the male–female gap was closing in more recent cohorts. The period of risk for drug initiation also appears to be lengthening longer into adulthood among more recent cohorts. Associations with sociodemographic variables were consistent across countries, as were the curves of incidence of lifetime use.

Conclusions

Globally, drug use is not distributed evenly and is not simply related to drug policy, since countries with stringent user-level illegal drug policies did not have lower levels of use than countries with liberal ones. Sex differences were consistently documented, but are decreasing in more recent cohorts, who also have higher levels of illegal drug use and extensions in the period of risk for initiation.     

Estimating treatment coverage for people with substance use disorders: an analysis of data from the World Mental Health Surveys

Substance use is a major cause of disability globally. This has been recognized in the recent United Nations Sustainable Development Goals (SDGs), in which treatment coverage for substance use disorders is identified as one of the indicators. There have been no estimates of this treatment coverage cross‐nationally, making it difficult to know what is the baseline for that SDG target. Here we report data from the World Health Organization (WHO)'s World Mental Health Surveys (WMHS), based on representative community household surveys in 26 countries. We assessed the 12‐month prevalence of substance use disorders (alcohol or drug abuse/dependence); the proportion of people with these disorders who were aware that they needed treatment and who wished to receive care; the proportion of those seeking care who received it; and the proportion of such treatment that met minimal standards for treatment quality (“minimally adequate treatment”). Among the 70,880 participants, 2.6% met 12‐month criteria for substance use disorders; the prevalence was higher in upper‐middle income (3.3%) than in high‐income (2.6%) and low/lower‐middle income (2.0%) countries. Overall, 39.1% of those with 12‐month substance use disorders recognized a treatment need; this recognition was more common in high‐income (43.1%) than in upper‐middle (35.6%) and low/lower‐middle income (31.5%) countries. Among those who recognized treatment need, 61.3% made at least one visit to a service provider, and 29.5% of the latter received minimally adequate treatment exposure (35.3% in high, 20.3% in upper‐middle, and 8.6% in low/lower‐middle income countries). Overall, only 7.1% of those with past‐year substance use disorders received minimally adequate treatment: 10.3% in high income, 4.3% in upper‐middle income and 1.0% in low/lower‐middle income countries. These data suggest that only a small minority of people with substance use disorders receive even minimally adequate treatment. At least three barriers are involved: awareness/perceived treatment need, accessing treatment once a need is recognized, and compliance (on the part of both provider and client) to obtain adequate treatment. Various factors are likely to be involved in each of these three barriers, all of which need to be addressed to improve treatment coverage of substance use disorders. These data provide a baseline for the global monitoring of progress of treatment coverage for these disorders as an indicator within the SDGs.     

N-terminal myristoylation regulates calcium-induced conformational changes in neuronal calcium sensor-1

Neuronal calcium sensor-1 (NCS-1), a Ca(2+)-binding protein, plays an important role in the modulation of neurotransmitter release and phosphatidylinositol signaling pathway. It is known that the physiological activity of NCS-1 is governed by its myristoylation. Here, we present the role of myristoylation of NSC-1 in governing Ca(2+) binding and Ca(2+)-induced conformational changes in NCS-1 as compared with the role in the nonmyristoylated protein. The (45)Ca binding and isothermal titration calorimetric data show that myristoylation increases the degree of cooperativity; thus, the myristoylated NCS-1 binds Ca(2+) more strongly (with three Ca(2+) binding sites) than the non-myristoylated one (with two Ca(2+) binding sites). Both forms of protein show different conformational features in far-UV CD when titrated with Ca(2+). Large conformational changes were seen in the near-UV CD with more changes in the case of nonmyristoylated protein than the myristoylated one. Although the changes in the far-UV CD upon Ca(2+) binding were not seen in E120Q mutant (disabling EF-hand 3), the near-UV CD changes in conformation also were not influenced by this mutation. The difference in the binding affinity of myristoylated and non-myristoylated proteins to Ca(2+) also was reflected by Trp fluorescence. Collisional quenching by iodide showed more inaccessibility of the fluorophore in the myristoylated protein. Mg(2+)-induced changes in near-UV CD are different from Ca(2+)-induced changes, indicating ion selectivity. 8-Anilino-1-naphthalene sulfonic acid binding data showed solvation of the myristoyl group in the presence of Ca(2+), which could be attributed to the myristoyl-dependent conformational changes in NCS-1. These results suggest that myristoylation influences the protein conformation and Ca(2+) binding, which might be crucial for its physiological functions.     

The cohesion protein ORD is required for homologue bias during meiotic recombination

During meiosis, sister chromatid cohesion is required for normal levels of homologous recombination, although how cohesion regulates exchange is not understood. Null mutations in orientation disruptor (ord) ablate arm and centromeric cohesion during Drosophila meiosis and severely reduce homologous crossovers in mutant oocytes. We show that ORD protein localizes along oocyte chromosomes during the stages in which recombination occurs. Although synaptonemal complex (SC) components initially associate with synapsed homologues in ord mutants, their localization is severely disrupted during pachytene progression, and normal tripartite SC is not visible by electron microscopy. In ord germaria, meiotic double strand breaks appear and disappear with frequency and timing indistinguishable from wild type. However, Ring chromosome recovery is dramatically reduced in ord oocytes compared with wild type, which is consistent with the model that defects in meiotic cohesion remove the constraints that normally limit recombination between sisters. We conclude that ORD activity suppresses sister chromatid exchange and stimulates inter-homologue crossovers, thereby promoting homologue bias during meiotic recombination in Drosophila.