Glucagon-like peptide-1 analog, liraglutide, regulates Sertoli cell energy metabolism.

Liraglutide, an analog of the incretin hormone glucagon-like peptide 1 (GLP-1), is widely used for obesity and type 2 diabetes treatment. However, there is scarce information about its effects on testicular function. Within the testis, Sertoli cells (SCs) provide nutritional support for germ cells; they metabolize glucose to lactate, which is delivered to germ cells to be used as a preferred energy substrate. Besides, SCs use fatty acids (FAs) as an energy source and store them as triacylglycerols (TAGs) within lipid droplets (LDs), which serve as an important energy reserve. In the present study, 20-day-old rat SC cultures were used to assess whether liraglutide affects their metabolic functions related to nutritional support and lipid storage. The results show that liraglutide does not modify glucose consumption or lactate production. However, it increases TAG levels and LD content. These effects are accompanied by an increase in the mRNA levels of the fatty acid transporter FAT/CD36, glycerol-3-phosphate-acyltransferase 3, and perilipins 1 and 4. The participation of the cAMP/PKA signaling pathway was explored. We observed that H89 (a PKA inhibitor) decreases the LD upregulation elicited by liraglutide, and that dibutyryl cAMP increases LD content and the expression of related genes. In summary, liraglutide promotes lipid storage in SCs through the regulation of key regulatory genes involved in FA transport, TAG synthesis, and LD formation. Considering the importance of lipid storage in SC energetic homeostasis maintenance, we postulate that liraglutide might improve the overall energetic status of the seminiferous tubule.

Ketogenic diet administration later in life improves memory by modifying the synaptic cortical proteome via the PKA signaling pathway in aging mice.

Aging compromises brain function leading to cognitive decline. A cyclic ketogenic diet (KD) improves memory in aged mice after long-term administration; however, short-term effects later in life and the molecular mechanisms that govern such changes remain unclear. Here, we explore the impact of a short-term KD treatment starting at elderly stage on brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings reveal that KD improves working memory and hippocampal LTP. Furthermore, the synaptosome proteome of aged mice fed a KD long-term evidence changes predominantly at the presynaptic compartment associated to the protein kinase A (PKA) signaling pathway. These findings were corroborated in vivo by western blot analysis, with high BDNF abundance and PKA substrate phosphorylation. Overall, we show that a KD modifies brain function even when it is administered later in life and recapitulates molecular features of long-term administration, including the PKA signaling pathway, thus promoting synaptic plasticity at advanced age.

Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks.

BACKGROUND: The selection of Saccharomyces cerevisiae strains with higher alcohol tolerance can potentially increase the industrial production of ethanol fuel. However, the design of selection protocols to obtain bioethanol yeasts with higher alcohol tolerance poses the challenge of improving industrial strains that are already robust to high ethanol levels. Furthermore, yeasts subjected to mutagenesis and selection, or laboratory evolution, often present adaptation trade-offs wherein higher stress tolerance is attained at the expense of growth and fermentation performance. Although these undesirable side effects are often associated with acute selection regimes, the utility of using harsh ethanol treatments to obtain robust ethanologenic yeasts still has not been fully investigated. RESULTS: We conducted an adaptive laboratory evolution by challenging four populations (P1-P4) of the Brazilian bioethanol yeast, Saccharomyces cerevisiae PE-2_H4, through 68-82 cycles of 2-h ethanol shocks (19-30% v/v) and outgrowths. Colonies isolated from the final evolved populations (P1c-P4c) were subjected to whole-genome sequencing, revealing mutations in genes enriched for the cAMP/PKA and trehalose degradation pathways. Fitness analyses of the isolated clones P1c-P3c and reverse-engineered strains demonstrated that mutations were primarily selected for cell viability under ethanol stress, at the cost of decreased growth rates in cultures with or without ethanol. Under this selection regime for stress survival, the population P4 evolved a protective snowflake phenotype resulting from BUD3 disruption. Despite marked adaptation trade-offs, the combination of reverse-engineered mutations cyr1A1474T/usv1Δ conferred 5.46% higher fitness than the parental PE-2_H4 for propagation in 8% (v/v) ethanol, with only a 1.07% fitness cost in a culture medium without alcohol. The cyr1A1474T/usv1Δ strain and evolved P1c displayed robust fermentations of sugarcane molasses using cell recycling and sulfuric acid treatments, mimicking Brazilian bioethanol production. CONCLUSIONS: Our study combined genomic, mutational, and fitness analyses to understand the genetic underpinnings of yeast evolution to ethanol shocks. Although fitness analyses revealed that most evolved mutations impose a cost for cell propagation, combination of key mutations cyr1A1474T/usv1Δ endowed yeasts with higher tolerance for growth in the presence of ethanol. Moreover, alleles selected for acute stress survival comprising the P1c genotype conferred stress tolerance and optimal performance under conditions simulating the Brazilian industrial ethanol production.

CaMKIIα mediates spermidine-induced memory enhancement in rats: A potential involvement of PKA/CREB pathway.

Memory consolidation is associated with the regulation of protein kinases, which impact synaptic functions and promote synaptogenesis. The administration of spermidine (SPD) has been shown to modulate major protein kinases associated with memory improvement, including the Ca2+-dependent protein kinase (PKC) and cAMP-dependent protein kinase (PKA), key players in the cAMP response element-binding protein (CREB) activation. Nevertheless, the initial mechanism underlying SPD-mediated memory consolidation remains unknown, as we hypothesize a potential involvement of the memory consolidation precursor, Ca2+/calmodulin-dependent protein kinase II-α (CaMKIIα), in this process. Based on this, our study aimed to investigate potential interactions among PKC, PKA, and CREB activation, mediated by CaMKIIα activation, in order to elucidate the SPD memory consolidation pathway. Our findings suggest that the post-training administration of the CaMKII inhibitor, KN-62 (0.25 nmol, intrahippocampal), prevented the memory enhancement induced by SPD (0.2 nmol, intrahippocampal) in the inhibitory avoidance task. Through western immunoblotting, we observed that phosphorylation of CaMKIIα in the hippocampus was facilitated 15 min after intrahippocampal SPD administration, resulting in the activation of PKA and CREB, 180 min after infusion, suggesting a possible sequential mechanism, since SPD with KN-62 infusion leads to a downregulation in CaMKIIα/PKA/CREB pathway. However, KN-62 does not alter the memory-facilitating effect of SPD on PKC, possibly demonstrating a parallel cascade in memory acquisition via PKA, without modulating CAMKIIα. These results suggest that memory enhancement induced by SPD administration involves crosstalk between CaMKIIα and PKA/CREB, with no PKC interaction.

The cAMP-PKA signalling crosstalks with CWI and HOG-MAPK pathways in yeast cell response to osmotic and thermal stress.

The yeast Saccharomyces cerevisiae is widely used in food and non-food industries. During industrial fermentation yeast strains are exposed to fluctuations in oxygen concentration, osmotic pressure, pH, ethanol concentration, nutrient availability and temperature. Fermentation performance depends on the ability of the yeast strains to adapt to these changes. Suboptimal conditions trigger responses to the external stimuli to allow homeostasis to be maintained. Stress-specific signalling pathways are activated to coordinate changes in transcription, translation, protein function, and metabolic fluxes while a transient arrest of growth and cell cycle progression occur. cAMP-PKA, HOG-MAPK and CWI signalling pathways are turned on during stress response. Comprehension of the mechanisms involved in the responses and in the adaptation to these stresses during fermentation is key to improving this industrial process. The scope of this review is to outline the advancement of knowledge about the cAMP-PKA signalling and the crosstalk of this pathway with the CWI and HOG-MAPK cascades in response to the environmental challenges heat and hyperosmotic stress.

A versatile kinase mobility shift assay (KiMSA) for PKA analysis and cyclic AMP detection in sperm physiology (and beyond).

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-32P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

Modulation of D3R Splicing, Signaling, and Expression by D1R through PKA→PTB Phosphorylation.

The D1R and D3R receptors functionally and synergistically interact in striatonigral neurons. Dopaminergic denervation turns this interaction antagonistic, which is correlated with a decrement in D3nf isoform and an increment in D3R membranal expression. The mechanisms of such changes in D3R are attributed to the dysregulation of the expression of their isoforms. The cause and mechanism of this phenomenon remain unknown. Dopaminergic denervation produces a decrement in D1R and PKA activity; we propose that the lack of phosphorylation of PTB (regulator of alternative splicing) by PKA produces the dysregulation of D3R splicing and changes D3R functionality. By using in silico analysis, we found that D3R mRNA has motifs for PTB binding and, by RIP, co-precipitates with PTB. Moreover, D1R activation via PKA promotes PTB phosphorylation. Acute and 5-day D1R blockade decreases the expression of D3nf mRNA. The 5-day treatment reduces D3R, D3nf, and PTB protein in the cytoplasm and increases D3R in the membrane and PTB in the nucleus. Finally, the blockade of D1R mimics the effect of dopaminergic denervation in D1R and D3R signaling. Thus, our data indicate that through PKA→PTB, D1R modulates D3R splicing, expression, and signaling, which are altered during D1R blockade or the lack of stimulation in dopaminergic denervation.

13 C NMR as an analytical tool for the detection of carbonic acid and pKa determination.

NMR spectroscopy has become a standard technique in studies both on carbon capture and storage. 13 C NMR allows the detection of two peaks for carbonated aqueous samples: one for CO2(aq) and another one for the species H2 CO3 , HCO3 - , and CO3 2- -herein collectively named Hx CO3 x-2 . The chemical shift of this second peak depends on the molar fraction of the three species in equilibrium and has been used to assess the equilibrium between HCO3 - and CO3 2- . The detection of H2 CO3 at low pH solutions is hindered, because of the concurrent liberation of CO2 when the medium is acidified. Herein, a valved NMR tube facilitates the detection of the Hx CO3 x-2 peak across a wide pH range, even at pH 1.8 where the dominant species is H2 CO3 . The method employed the formation of frozen layers of NaH13 CO3 and acid solutions within the tube, which are mixed as the tube reaches room temperature. At this point, the tube is already securely sealed, preventing any loss of CO2 to the atmosphere. A spectrophotometry approach allowed the measurement of the actual pH inside the pressurized NMR tube. The chemical shift for H2 CO3 was determined as 160.33 ± 0.03 ppm, which is in good agreement with value obtained by DFT calculations combined with Car-Parrinello molecular dynamics. The H2 CO3 pKa value determined by the present method was 3.41 ± 0.03, for 15% D2 O aqueous medium and 0.8 mol/L ionic strength. The proposed method can be extended to studies about analogs such as alkyl carbonic and carbamic acids.

Transcription Factors Tec1 and Tec2 Play Key Roles in the Hyphal Growth and Virulence of Mucor lusitanicus Through Increased Mitochondrial Oxidative Metabolism.

Mucormycosis is a lethal and difficult-to-treat fungal infection caused by fungi of the order Mucorales. Mucor lusitanicus, a member of Mucorales, is commonly used as a model to understand disease pathogenesis. However, transcriptional control of hyphal growth and virulence in Mucorales is poorly understood. This study aimed to investigate the role of Tec proteins, which belong to the TEA/ATTS transcription factor family, in the hyphal development and virulence of M. lusitanicus. Unlike in the genome of Ascomycetes and Basidiomycetes, which have a single Tec homologue, in the genome of Mucorales, two Tec homologues, Tec1 and Tec2, were found, except in that of Phycomyces blakesleeanus, with only one Tec homologue. tec1 and tec2 overexpression in M. lusitanicus increased mycelial growth, mitochondrial content and activity, expression of the rhizoferrin synthetase-encoding gene rfs, and virulence in nematodes and wax moth larvae but decreased cAMP levels and protein kinase A (PKA) activity. Furthermore, tec1- and tec2-overexpressing strains required adequate mitochondrial metabolism to promote the virulent phenotype. The heterotrimeric G beta subunit 1-encoding gene deletant strain (Δgpb1) increased cAMP-PKA activity, downregulation of both tec genes, decreased both virulence and hyphal development, but tec1 and tec2 overexpression restored these defects. Overexpression of allele-mutated variants of Tec1(S332A) and Tec2(S168A) in the putative phosphorylation sites for PKA increased both virulence and hyphal growth of Δgpb1. These findings suggest that Tec homologues promote mycelial development and virulence by enhancing mitochondrial metabolism and rhizoferrin accumulation, providing new information for the rational control of the virulent phenotype of M. lusitanicus.

Aging: silencing the PKA and AkT/PKB signaling pathways alters the antioxidant capacity of resveratrol.

One of the theories related to aging is the increase in oxidative stress. Given this, the objective of the study is to evaluate the cellular mechanisms responsible for the resveratrol antioxidant effect on leukocytes from donors aged between 20 and 80 years old. For this, leukocytes from donors of three age groups (20-39, 40-59 and 60-80) were isolated. Image-iT™LIVE Green Reactive Oxygen Species (ROS) Kit was used. Reactive Nitrogen Species (RNS) analysis was performed by measuring nitric oxide and peroxynitrite. The PKA, Akt/PKB and p38-MAPK were evaluated by chemiluminescence. The statistical analysis between age and treatments were performed by Pearson correlation (*p < 0.05). It was possible to observe the antioxidant effect of resveratrol in all age groups. The correlation results show loss of resveratrol effect in decreasing ROS in leukocytes from older donors. We observed an active antioxidant effect of p38-MAPK in all ages, with resveratrol acting on it. The PKA and Akt/PKB were active in leukocytes from donors aged 20-59. In cells from donors older than 60, these pathways are silenced, and an effect is also not observed in cells treated with resveratrol. Therefore, resveratrol showed antioxidant effect in all age, although it was more pronounced in leukocytes from younger. One of resveratrol's mechanisms is due to the activation of the PKA and Akt/PKB, which were activated in younger donor cells.

Impact of interorganelle coordination between the conventional early secretory pathway and autophagy in cellular homeostasis and stress response.

The conventional early secretory pathway and autophagy are two essential interconnected cellular processes that are crucial for maintaining cellular homeostasis. The conventional secretory pathway is an anabolic cellular process synthesizing and delivering proteins to distinct locations, including different organelles, the plasma membrane, and the extracellular media. On the other hand, autophagy is a catabolic cellular process that engulfs damaged organelles and aberrant cytosolic constituents into the double autophagosome membrane. After fusion with the lysosome and autolysosome formation, this process triggers digestion and recycling. A growing list of evidence indicates that these anabolic and catabolic processes are mutually regulated. While knowledge about the molecular actors involved in the coordination and functional cooperation between these two processes has increased over time, the mechanisms are still poorly understood. This review article summarized and discussed the most relevant evidence about the key molecular players implicated in the interorganelle crosstalk between the early secretory pathway and autophagy under normal and stressful conditions.

Dissociation Mode of the O-H Bond in Betanidin, pKa-Clusterization Prediction, and Molecular Interactions via Shape Theory and DFT Methods.

Betanidin (Bd) is a nitrogenous metabolite with significant bioactive potential influenced by pH. Its free radical scavenging activity and deprotonation pathway are crucial to studying its physicochemical properties. Motivated by the published discrepancies about the best deprotonation routes in Bd, this work explores all possible pathways for proton extractions on that molecule, by using the direct approach method based on pKa. The complete space of exploration is supported by a linear relation with constant slope, where the pKa is written in terms of the associated deprotonated molecule energy. The deprotonation rounds 1, …, 6 define groups of parallel linear models with constant slope. The intercepts of the models just depend on the protonated energy for each round, and then the pKa can be trivially ordered and explained by the energy. We use the direct approximation method to obtain the value of pKa. We predict all possible outcomes based on a linear model of the energy and some related verified assumptions. We also include a new measure of similarity or dissimilarity between the protonated and deprotonated molecules, via a geometric-chemical descriptor called the Riemann-Mulliken distance (RMD). The RMD considers the cartesian coordinates of the atoms, the atomic mass, and the Mulliken charges. After exploring the complete set of permutations, we show that the successive deprotonation process does not inherit the local energy minimum and that the commutativity of the paths does not hold either. The resulting clusterization of pKa can be explained by the local acid and basic groups of the BD, and the successive deprotonation can be predicted by using the chemical explained linear models, which can avoid unnecessary optimizations. Another part of the research uses our own algorithm based on shape theory to determine the protein's active site automatically, and molecular dynamics confirmed the results of the molecular docking of Bd in protonated and anionic form with the enzyme aldose reductase (AR). Also, we calculate the descriptors associated with the SET and SPLET mechanisms.

Role of Protein Kinase A Activation in the Immune System with an Emphasis on Lipopolysaccharide-Responsive and Beige-like Anchor Protein in B Cells.

Cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous enzymatic complex that is involved in a broad spectrum of intracellular receptor signaling. The activity of PKA depends on A-kinase anchoring proteins (AKAPs) that attach to PKAs close to their substrates to control signaling. Although the relevance of PKA-AKAP signaling in the immune system is evident in T cells, its relevance in B and other immune cells remains relatively unclear. In the last decade, lipopolysaccharide-responsive and beige-like anchor protein (LRBA) has emerged as an AKAP that is ubiquitously expressed in B and T cells, specifically after activation. A deficiency of LRBA leads to immune dysregulation and immunodeficiency. The cellular mechanisms regulated by LRBA have not yet been investigated. Therefore, this review summarizes the functions of PKA in immunity and provides the most recent information regarding LRBA deficiency to deepen our understanding of immune regulation and immunological diseases.

Predicting octanol/water partition coefficients and pKa for the SAMPL7 challenge using the SM12, SM8 and SMD solvation models.

Blind predictions of octanol/water partition coefficients and pKa at 298.15 K for 22 drug-like compounds were made for the SAMPL7 challenge. Octanol/water partition coefficients were predicted from solvation free energies computed using electronic structure calculations with the SM12, SM8 and SMD solvation models. Within these calculations we compared the use of gas- and solution-phase optimized geometries of the solute. Based on these calculations we found that in general the use of solution phase-optimized geometries increases the affinity of the solutes for water as compared to octanol, with the use of gas-phase optimized geometries resulting in the better agreement with experiment. The pKa is computed using the direct approach, scaled solvent-accessible surface model, and the inclusion of an explicit water molecule, where the latter two methods have previously been shown to offer improved predictions as compared to the direct approach. We find that the use of an explicit water molecule provides superior predictions, and that the predicted macroscopic pKa is sensitive to the employed microstates.

IL-33 and the PKA Pathway Regulate ILC2 Populations Expressing IL-9 and ST2.

Type 2 Innate lymphoid cells (ILC2s) are tissue-resident immune cells activated by epithelial-derived alarmins upon tissue damage. They regulate immunity against helminth parasites and allergies by expressing type 2 immune response cytokines including IL-9, known to be critical for inducing and potentiating the immune response in such context. Although ILC2s are reported to be the main source of IL-9 in mice during N. brasiliensis infection, the mechanisms that regulate the expression of IL-9 in these cells are yet to be described. Recent studies have shown that in addition to cytokines, multiple molecules can differentially modulate the functions of ILC2s in various contexts both in vitro and in vivo. Among these stimuli are lipid mediators and neuropeptides, which activate the PKA pathway and have been associated with the regulation of type 2 immune cytokines. In this work we found that ILC2s in mice infected with N. brasiliensis can be classified into different groups based on the expression of IL-9 and ST2. These distinct populations were distributed in the lung and the small intestine. Through the development of an in vitro culture system, we sought to determine the stimuli that regulate the expression of these markers in ILC2s. We identified the alarmin IL-33 as being a key player for increased IL-9 expression. Additionally, we found the PKA pathway to be a dual regulator of ILC2 cells, working synergistically with IL-33 to enhance IL-9 production and capable of modulating proliferation and the expression of ILC2 markers. These data provide further evidence of a high heterogeneity between ILC2 subsets in a context dependent manner and calls for careful consideration when choosing the markers to identify these cells in vivo. Distinguishing ILC2 subsets and dissecting their mechanisms of activation is critical for a deeper understanding of the biology of these cells, allowing their manipulation for therapeutic purposes.

Bone Progenitors Pull the Strings on the Early Metabolic Rewiring Occurring in Prostate Cancer Cells.

Metastatic prostate cancer (PCa) cells soiling in the bone require a metabolic adaptation. Here, we identified the metabolic genes fueling the seeding of PCa in the bone niche. Using a transwell co-culture system of PCa (PC3) and bone progenitor cells (MC3T3 or Raw264.7), we assessed the transcriptome of PC3 cells modulated by soluble factors released from bone precursors. In a Principal Component Analysis using transcriptomic data from human PCa samples (GSE74685), the altered metabolic genes found in vitro were able to stratify PCa patients in two defined groups: primary PCa and bone metastasis, confirmed by an unsupervised clustering analysis. Thus, the early transcriptional metabolic profile triggered in the in vitro model has a clinical correlate in human bone metastatic samples. Further, the expression levels of five metabolic genes (VDR, PPARA, SLC16A1, GPX1 and PAPSS2) were independent risk-predictors of death in the SU2C-PCF dataset and a risk score model built using this lipid-associated signature was able to discriminate a subgroup of bone metastatic PCa patients with a 23-fold higher risk of death. This signature was validated in a PDX pre-clinical model when comparing MDA-PCa-183 growing intrafemorally vs. subcutaneously, and appears to be under the regulatory control of the Protein Kinase A (PKA) signaling pathway. Secretome analyses of conditioned media showcased fibronectin and type-1 collagen as critical bone-secreted factors that could regulate tumoral PKA. Overall, we identified a novel lipid gene signature, driving PCa aggressive metastatic disease pointing to PKA as a potential hub to halt progression.

Virtual Prospection of Marine Cyclopeptides as Therapeutics by Means of Conceptual DFT and Computational ADMET.

Bioactive peptides are chemical compounds created through the covalent bonding of amino acids, known as amide or peptide bonds. Due to their unusual chemistry and various biological effects, marine bioactive peptides have garnered considerable research. The effectiveness of a bioactive marine peptide is attributed to its structural features, such as amino acid content and sequence, which vary depending on the degree of action. Cyclic peptides combine several favorable properties such as good binding affinity, target selectivity and low toxicity that render them an attractive modality for the development of therapeutics. The apratoxins are a class of molecules formed by a series of cyclic depsipeptides with potent cytotoxic activities. The objective of this research is to pursue a computational prospection of the molecular structures and properties of several cylopeptides of marine origin with potential therapeutic applications. The methodology will be based on the determination of the chemical reactivity descriptors of the studied molecules through the consideration of the Conceptual DFT model and validation of a particular model chemistry, MN12SX/Def2TZVP/H2O. These studies will be complemented by a determination of the pharmacokinetics and ADMET parameters by resorting to certain cheminformatics tools.

Integration of In Silico, In Vitro and In Situ Tools for the Preformulation and Characterization of a Novel Cardio-Neuroprotective Compound during the Early Stages of Drug Development.

The main aim of this work is the biopharmaceutical characterization of a new hybrid benzodiazepine-dihydropyridine derivative, JM-20, derived with potent anti-ischemic and neuroprotective effects. In this study, the pKa and the pH-solubility profile were experimentally determined. Additionally, effective intestinal permeability was measured using three in vitro epithelial cell lines (MDCK, MDCK-MDR1 and Caco-2) and an in situ closed-loop intestinal perfusion technique. The results indicate that JM-20 is more soluble at acidic pH (9.18 ± 0.16); however, the Dose number (Do) was greater than 1, suggesting that it is a low-solubility compound. The permeability values obtained with in vitro cell lines as well as with the in situ perfusion method show that JM-20 is a highly permeable compound (Caco-2 value 3.8 × 10-5). The presence of an absorption carrier-mediated transport mechanism was also demonstrated, as well as the efflux effect of P-glycoprotein on the permeability values. Finally, JM-20 was provisionally classified as class 2 according to the biopharmaceutical classification system (BCS) due to its high intestinal permeability and low solubility. The potential good oral absorption of this compound could be limited by its solubility.

Heat stress regulates the expression of TPK1 gene at transcriptional and post-transcriptional levels in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae cAMP regulates different cellular processes through PKA. The specificity of the response of the cAMP-PKA pathway is highly regulated. Here we address the mechanism through which the cAMP-PKA pathway mediates its response to heat shock and thermal adaptation in yeast. PKA holoenzyme is composed of a regulatory subunit dimer (Bcy1) and two catalytic subunits (Tpk1, Tpk2, or Tpk3). PKA subunits are differentially expressed under certain growth conditions. Here we demonstrate the increased abundance and half-life of TPK1 mRNA and the assembly of this mRNA in cytoplasmic foci during heat shock at 37 °C. The resistance of the foci to cycloheximide-induced disassembly along with the polysome profiling analysis suggest that TPK1 mRNA is impaired for entry into translation. TPK1 expression was also evaluated during a recurrent heat shock and thermal adaptation. Tpk1 protein level is significantly increased during the recovery periods. The crosstalk of cAMP-PKA pathway and CWI signalling was also studied. Wsc3 sensor and some components of the CWI pathway are necessary for the TPK1 expression upon heat shock. The assembly in foci upon thermal stress depends on Wsc3. Tpk1 expression is lower in a wsc3∆ mutant than in WT strain during thermal adaptation and thus the PKA levels are also lower. An increase in Tpk1 abundance in the PKA holoenzyme in response to heat shock is presented, suggesting that a recurrent stress enhanced the fitness for the coming favourable conditions. Therefore, the regulation of TPK1 expression by thermal stress contributes to the specificity of cAMP-PKA signalling.

Lithium modulates multiple tau kinases with distinct effects in cortical and hippocampal neurons according to concentration ranges.

The hyperphosphorylation of tau is a central mechanism in the pathogenesis of Alzheimer's disease (AD). Lithium is a potent inhibitor of glycogen synthase kinase-3beta (GSK3ß), the most important tau kinase in neurons, and may also affect tau phosphorylation by modifying the expression and/or activity of other kinases, such as protein kinase A (PKA), Akt (PKB), and calcium calmodulin kinase-II (CaMKII). The aim of the present study is to determine the effect of chronic lithium treatment on the protein expression of tau and its major kinases in cortical and hippocampal neurons, at distinct working concentrations. Primary cultures of cortical and hippocampal neurons were treated with sub-therapeutic (0.02 mM and 0.2 mM) and therapeutic (2 mM) concentrations of lithium for 7 days. Protein expression of tau and tau-kinases was determined by immunoblotting. An indirect estimate of GSK3ß activity was determined by the GSK3ß ratio (rGSKß). Statistically significant increments in the protein expression of tau and CaMKII were observed both in cortical and hippocampal neurons treated with subtherapeutic doses of lithium. GSK3ß activity was increased in cortical, but decreased in hippocampal neurons. Distinct patterns of changes in the expression of the remaining tau tau-kinases were observed: in cortical neurons, lithium treatment was associated with consistent decrements in Akt and PKA, whereas hippocampal neurons displayed increased protein expression of Akt and decreased PKA. Our results suggest that chronic lithium treatment may yield distinct biological effects depending on the concentration range, with regional specificity. We further suggest that hippocampal neurons may be more sensitive to the effect of lithium, presenting with changes in the expression of tau-related proteins at subtherapeutic doses, which may not be mirrored by the effects observed in cortical neurons.