Palmitoylation targets AKAP79 protein to lipid rafts and promotes its regulation of calcium-sensitive adenylyl cyclase type 8

PKA anchoring proteins (AKAPs) optimize the efficiency of cAMP signaling by clustering interacting partners. Recently, AKAP79 has been reported to directly bind to adenylyl cyclase type 8 (AC8) and to regulate its responsiveness to store-operated Ca(2+) entry (SOCE). Although AKAP79 is well targeted to the plasma membrane via phospholipid associations with three N-terminal polybasic regions, recent studies suggest that AKAP79 also has the potential to be palmitoylated, which may specifically allow it to target the lipid rafts where AC8 resides and is regulated by SOCE. In this study, we have addressed the role of palmitoylation of AKAP79 using a combination of pharmacological, mutagenesis, and cell biological approaches. We reveal that AKAP79 is palmitoylated via two cysteines in its N-terminal region. This palmitoylation plays a key role in targeting the AKAP to lipid rafts in HEK-293 cells. Mutation of the two critical cysteines results in exclusion of AKAP79 from lipid rafts and alterations in its membrane diffusion behavior. This is accompanied by a loss of the ability of AKAP79 to regulate SOCE-dependent AC8 activity in intact cells and decreased PKA-dependent phosphorylation of raft proteins, including AC8. We conclude that palmitoylation plays a key role in the targeting and action of AKAP79. This novel property of AKAP79 adds an unexpected regulatory and targeting option for AKAPs, which may be exploited in the cellular context.     

Effects of hypoxia on muscle protein synthesis and anabolic signaling at rest and in response to acute resistance exercise

Chronic reductions in tissue O(2) tension (hypoxia) are associated with muscle atrophy and blunted hypertrophic responses to resistance exercise (RE) training. However, the effect of hypoxia on muscle protein synthesis (MPS) at rest and after RE is unknown. In a crossover study, seven healthy men (21.4 ± 0.7 yr) performed unilateral leg RE (6 × 8 repetitions at 70% 1-repetition maximum) under normoxic (20.9% inspired O(2)) and normobaric hypoxic (12% inspired O(2) for 3.5 h) postabsorptive conditions. Immediately after RE the rested leg was biopsied, and a primed continuous infusion of [1,2-(13)C(2)]leucine was maintained for 2.5 h before final biopsies from both legs to measure tracer incorporation and signaling responses (i.e., ribosomal S6 kinase 1). After 3.5 h of hypoxia, MPS was not different from normoxia in the rested leg (normoxia 0.033 ± 0.016 vs. hypoxia 0.043 ± 0.016%/h). MPS increased significantly from baseline 2.5 h after RE in normoxia (0.033 ± 0.016 vs. 0.104 ± 0.038%/h) but not hypoxia (0.043 ± 0.016 vs. 0.060 ± 0.063%/h). A significant linear relationship existed between MPS 2.5 h after RE in hypoxia and mean arterial blood O(2) saturation during hypoxia (r(2) = 0.49, P = 0.04). Phosphorylation of p70S6K(Thr389) remained unchanged in hypoxia at rest but increased after RE in both normoxia and hypoxia (2.6 ± 1.2-fold and 3.4 ± 1.1-fold, respectively). Concentrations of the hypoxia-responsive mTOR inhibitor regulated in development and DNA damage-1 were unaltered by hypoxia or RE. We conclude that normobaric hypoxia does not reduce MPS over 3.5 h at rest but blunts the increased MPS response to acute RE to a degree dependent on extant SpO(2).     

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.     

Migration patterns and navigation cues of Atlantic salmon post-smolts migrating from 12 rivers through the coastal zones around the Irish Sea

The freshwater phase of the first seaward migration of juvenile Atlantic salmon (Salmo salar) is relatively well understood when compared with our understanding of the marine phase of their migration. In 2021, 1008 wild and 60 ranched Atlantic salmon smolts were tagged with acoustic transmitters in 12 rivers in England, Scotland, Northern Ireland and Ireland. Large marine receiver arrays were deployed in the Irish Sea at two locations: at the transition of the Irish Sea into the North Atlantic between Ireland and Scotland, and between southern Scotland and Northern Ireland, to examine the early phase of the marine migration of Atlantic salmon smolts. After leaving their natal rivers' post-smolt migration through the Irish Sea was rapid with minimum speeds ranging from 14.03 to 38.56 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal river, to 9.69–39.94 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal estuary. Population minimum migration success through the study area was strongly correlated with the distance of travel, populations further away from the point of entry to the open North Atlantic exhibited lower migration success. Post-smolts from different populations experienced different water temperatures on entering the North Atlantic. This was largely driven by the timing of their migration and may have significant consequences for feeding and ultimately survivorship. The influence of water currents on post-smolt movement was investigated using data from previously constructed numerical hydrodynamic models. Modeled water current data in the northern Irish Sea showed that post-smolts had a strong preference for migrating when the current direction was at around 283° (west-north-west) but did not migrate when exposed to strong currents in other directions. This is the most favorable direction for onward passage from the Irish Sea to the continental shelf edge current, a known accumulation point for migrating post-smolts. These results strongly indicate that post-smolts migrating through the coastal marine environment are: (1) not simply migrating by current following (2) engage in active directional swimming (3) have an intrinsic sense of their migration direction and (4) can use cues other than water current direction to orientate during this part of their migration.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

Global diversity and oceanic divergence of humpback whales (Megaptera novaeangliae)

Humpback whales (Megaptera novaeangliae) annually undertake the longest migrations between seasonal feeding and breeding grounds of any mammal. Despite this dispersal potential, discontinuous seasonal distributions and migratory patterns suggest that humpbacks form discrete regional populations within each ocean. To better understand the worldwide population history of humpbacks, and the interplay of this species with the oceanic environment through geological time, we assembled mitochondrial DNA control region sequences representing approximately 2700 individuals (465 bp, 219 haplotypes) and eight nuclear intronic sequences representing approximately 70 individuals (3700 bp, 140 alleles) from the North Pacific, North Atlantic and Southern Hemisphere. Bayesian divergence time reconstructions date the origin of humpback mtDNA lineages to the Pleistocene (880 ka, 95% posterior intervals 550–1320 ka) and estimate radiation of current Northern Hemisphere lineages between 50 and 200 ka, indicating colonization of the northern oceans prior to the Last Glacial Maximum. Coalescent analyses reveal restricted gene flow between ocean basins, with long-term migration rates (individual migrants per generation) of less than 3.3 for mtDNA and less than 2 for nuclear genomic DNA. Genetic evidence suggests that humpbacks in the North Pacific, North Atlantic and Southern Hemisphere are on independent evolutionary trajectories, supporting taxonomic revision of M. novaeangliae to three subspecies.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.     

γδT Cells Are Prevalent in the Proximal Aorta and Drive Nascent Atherosclerotic Lesion Progression and Neutrophilia in Hypercholesterolemic Mice

Unique innate immunity-linked γδT cells have been seen in early human artery lesions, but their role in lesion development has received little attention. Here we investigated whether γδT cells modulate atherogenesis in apolipoprotein E-deficient (ApoE KO) mice. We found that γδT cell numbers were markedly increased in the proximal aorta of ApoE-deficient vs. wild-type mice during early atherogenesis, particularly in the aortic root and arch, where they comprised most of the T cells and lesion progression is most rapid. γδT cells infiltrated intimal lesions in ApoE KO mice, but only the adventitia in wild-type mice, and were more prevalent than CD4⁺ T cells in early nascent lesions, as evaluated by en face confocal microscopy. These aortic γδT cells produced IL-17, but not IFN-γ, analyzed by ex vivo FACS. Furthermore, aortic arch lipid accumulation correlated strongly with abundance of IL-17-expressing splenic γδT cells in individual ApoE KO mice. To investigate the role of these γδT cells in early atherogenesis, we analyzed ApoE/γδT double knockout (DKO) compared to ApoE KO mice. We observed reduced early intimal lipid accumulation at sites of nascent lesion formation, both in chow-fed (by 40%) and Western diet-fed (by 44%) ApoE/γδT DKO mice. In addition, circulating neutrophils were drastically reduced in these DKO mice on Western diet, while expansion of inflammatory monocytes and splenic Th1 or Th17 lymphocytes was not affected. These data reveal, for the first time, a pathogenic role of γδT cells in early atherogenesis in ApoE KO mice, by mechanisms likely to involve their IL-17 production and induction of neutrophilia. Targeting γδT cells thus might offer therapeutic benefit in atherosclerosis or other inflammatory vascular diseases.     

ADH1B and ADH1C Genotype,Alcohol Consumption and Biomarkers of Liver Function: Findings from a Mendelian Randomization Study in 58,313 European Origin Danes

Background

The effect of alcohol consumption on liver function is difficult to determine because of reporting bias and potential residual confounding. Our aim was to determine this effect using genetic variants to proxy for the unbiased effect of alcohol.

Methods

We used variants in ADH1B and ADH1C genes as instrumental variables (IV) to estimate the causal effect of long-term alcohol consumption on alanine aminotransferase (ALT), γ-glutamyl-transferase (γ-GT), alkaline phosphatase (ALP), bilirubin and prothrombin action. Analyses were undertaken on 58,313 Danes (mean age 56).

Results

In both confounder adjusted multivariable and genetic-IV analyses greater alcohol consumption, amongst those who drank any alcohol, was associated with higher ALT [mean difference per doubling of alcohol consumption: 3.4% (95% CI: 3.1, 3.7) from multivariable analyses and 3.7% (−4.5, 11.9) from genetic-IV analyses] and γ-GT [8.2% (7.8, 8.5) and 6.8% (−2.8, 16.5)]. The point estimates from the two methods were very similar and statistically the results from the two methods were consistent with each other for effects with ALT and γ-GT (both p_diff>0.3). Results from the multivariable analyses suggested a weak inverse association of alcohol with ALP [−1.5% (−1.7, −1.3)], which differed from the strong positive effect found in genetic-IV analyses [11.6% (6.8, 16.4)] (p_diff<0.0001). In both multivariable and genetic-IV analyses associations with bilirubin and protrombin action were weak and close to the null.

Conclusions

Our results suggest that greater consumption of alcohol is related to poorer liver function as indicated by higher ALT, γ-GT and ALP, but not to clotting or bilirubin.