Intracellular cAMP Concentration Research Articles

The multidrug resistance protein (MRP) 4 is expressed and functionally active in isolated bladder from pig.

Multidrug resistance proteins type 4 (MRP4) and 5 (MRP5) play pivotal roles in the transport of cyclic nucleotides in various tissues. However, their specific functions within the lower urinary tract remain relatively unexplored. This study aimed to investigate the effect of pharmacological inhibition of MRPs on cyclic nucleotide signaling in isolated pig bladder. The relaxation responses of the bladder were assessed in the presence of the MRP inhibitor, MK571. The temporal changes in intra- and extracellular levels of cAMP and cGMP in stimulated tissues were determined by mass spectrometry. The gene (ABCC4) and protein (MRP4) expression were also determined. MK571 administration resulted in a modest relaxation effect of approximately 26% in carbachol-pre-contracted bladders. The relaxation induced by phosphodiesterase inhibitors such as cilostazol, tadalafil, and sildenafil was significantly potentiated in the presence of MK571. In contrast, no significant potentiation was observed in the relaxation induced by substances elevating cAMP levels or stimulators of soluble guanylate cyclase. Following forskolin stimulation, both intracellular and extracellular cAMP concentrations increased by approximately 15.8-fold and 12-fold, respectively. Similarly, stimulation with tadalafil + BAY 41-2272 resulted in roughly 8.2-fold and 3.4-fold increases in intracellular and extracellular cGMP concentrations, respectively. The presence of MK571 reduced only the extracellular levels of cGMP. This study reveals the presence and function of MRP4 transporters within the porcine bladder and paves the way for future research exploring the role of this transporter in both underactive and overactive bladder disorders.

Anti-hair loss effect of a shampoo containing caffeine and adenosine.

Hair loss is a widespread health problem that affects numerous individuals and is associated with age, lack of sleep, stress, endocrine problems, and other problems. Caffeine exerts various pharmacological effects, particularly after ingestion. The caffeine-induced inhibition of phosphodiesterases can increase intracellular cAMP concentrations, ultimately resulting in stimulatory effects on cell metabolism and proliferation. Hence, caffeine has been confirmed to inhibit hair loss caused by premature termination of the hair growth phase. Adenosine also improves hair loss by stimulating hair growth and thickening hair shafts. However, further empirical evidence is required to comprehensively assess the efficacy of hair loss treatment and prevention using a formulation of caffeine and adenosine in specific proportions in shampoos. This study aimed to evaluate a shampoo with caffeine and adenosine as a daily scalp care product for hair loss in 77 subjects aged 18-60 years. The overall and local hair densities were assessed using professional cameras and dermoscopes at different magnifications and distances. Five hairs that came off the participant's head were randomly selected to measure hair diameter. The self-assessment questionnaires were filled on third month of product use. The combination of caffeine and adenosine in the shampoo significantly enhanced hair density compared to that of the baseline. The results revealed a significant reduction in hair loss. The hair diameters of the subjects did not change significantly. Most of the participants (71.05%) were satisfied with their hair after using the product. Shampoos containing caffeine and adenosine have been demonstrated to exert therapeutic benefits for reducing hair loss.

Molecular characterization of GPR84 in domestic cats

G protein-coupled receptor 84 (GPR84) was cloned as an orphan receptor, and medium-chain fatty acids were then revealed as endogenous ligands. GPR84 is expressed in immune cells and is believed to protect liver function from lipotoxicity caused by overeating and high-fat diet intake. This study aimed to present the molecular characterization of GPR84 in domestic cats. The deduced amino acid sequence of the feline GPR84 shows high sequence homology (83–89 %) with the orthologues from other mammalians by cDNA cloning of feline GPR84. Remarkably high mRNA expression was observed in the bone marrow by Q-PCR analysis. The inhibition of intracellular cAMP concentration was observed in cells transfected with feline GPR84 and treated with medium-chain fatty acids. Immunostaining of GPR84 and free fatty acid receptor 2 (FFAR2)/GPR43 in the bone marrow, where high mRNA expression was observed, showed reactions in macrophages and myeloid cells. To clarify whether the receptor formed homo/hetero-merization, GPR84 and FFARs were analyzed using Nano-Luc binary technology and NanoLuc bioluminescence resonance energy transfer technologies, which revealed that GPR84 formed more heteromers with FFAR2 than homomers with each other. In addition, when GPR84 and FFAR2/GPR43 were cotransfected in the cell, their localization on the cell membrane was reduced compared with that when single receptors were transfected. These results indicated that GPR84 is a functional receptor protein that is expressed in cat tissues and may have a protein–protein interaction with FFAR2/GPR43 on the cell membrane.

Transcriptional and Metabolic Response of a Strain of Escherichia coli PTS- to a Perturbation of the Energetic Level by Modification of [ATP]/[ADP] Ratio.

The intracellular [ATP]/[ADP] ratio is crucial for Escherichia coli's cellular functions, impacting transport, phosphorylation, signaling, and stress responses. Overexpression of F1-ATPase genes in E. coli increases glucose consumption, lowers energy levels, and triggers transcriptional responses in central carbon metabolism genes, particularly glycolytic ones, enhancing carbon flux. In this contribution, we report the impact of the perturbation of the energetic level in a PTS- mutant of E. coli by modifying the [ATP]/[ADP] ratio by uncoupling the cytoplasmic activity of the F1 subunit of the ATP synthase. The disruption of [ATP]/[ADP] ratio in the evolved strain of E. coli PB12 (PTS-) was achieved by the expression of the atpAGD operon encoding the soluble portion of ATP synthase F1-ATPase (strain PB12AGD+). The analysis of the physiological and metabolic response of the PTS- strain to the ATP disruption was determined using RT-qPCR of 96 genes involved in glucose and acetate transport, glycolysis and gluconeogenesis, pentose phosphate pathway (PPP), TCA cycle and glyoxylate shunt, several anaplerotic, respiratory chain, and fermentative pathways genes, sigma factors, and global regulators. The apt mutant exhibited reduced growth despite increased glucose transport due to decreased energy levels. It heightened stress response capabilities under glucose-induced energetic starvation, suggesting that the carbon flux from glycolysis is distributed toward the pentose phosphate and the Entner-Duodoroff pathway with the concomitant. Increase acetate transport, production, and utilization in response to the reduction in the [ATP]/[ADP] ratio. Upregulation of several genes encoding the TCA cycle and the glyoxylate shunt as several respiratory genes indicates increased respiratory capabilities, coupled possibly with increased availability of electron donor compounds from the TCA cycle, as this mutant increased respiratory capability by 240% more than in the PB12. The reduction in the intracellular concentration of cAMP in the atp mutant resulted in a reduced number of upregulated genes compared to PB12, suggesting that the mutant remains a robust genetic background despite the severe disruption in its energetic level.

Effect of polymorphisms in the FAM151B gene on testis-related trait parameters in Hu sheep

The family with sequence similarity 151 member B (FAM151B) gene family is widely distributed in animals. This gene family encodes proteins belonging to the phosphodiesterase superfamily which plays a role in hydrolyzing intracellular second messengers, and affects the proliferation of Sertoli cells in the testis by degrading intracellular cAMP or cGMP content, thereby affecting the sperm production capacity of the testis. In this study, mutations in the FAM151B gene were screened in 491 Hu sheep to analyze the association among FAM151B gene polymorphisms testicular traits, and sperm-related parameters. A synonymous mutation (5: g.77976636 C>T), was detected in FAM151B, resulting in three genotypes, namely, CC, TT, and TC, with genotypic frequencies of 0.33, 0.05, and 0.62, respectively. This synonymous mutation was found to be significantly correlated with testicular weight, long testicular circumference, short testicular circumference, epididymal weight, and sperm count (p <0.05). In addition, individuals with CC genotypes had significant reproductive performance advantages compared with those with TC and TT genotypes. The current study shows that the FAM151B gene may play important roles in testicular development, and its SNPs are associated with testicular parameters, and sperm count, which provides important indicators for ram selection at early stage.

The regulatory effects of (p)ppGpp and indole on cAMP synthesis in Escherichia coli cells.

Bacterial stress adaptive response is formed due to changes in the cell gene expression profile in response to alterations in environmental conditions through the functioning of regulatory networks. The mutual influence of network signaling molecules represented by cells' natural metabolites, including indole and second messengers (p) ppGpp and cAMP, is hitherto not well understood, being the aim of this study. E. coli parent strain BW25141 ((p) ppGpp+) and deletion knockout BW25141ΔrelAΔspoT which is unable to synthesize (p)ppGpp ((p)ppGpp0) were cultivated in M9 medium supplemented with different glucose concentrations (5.6 and 22.2 mM) in the presence of tryptophan as a substrate for indole synthesis and in its absence. The glucose content was determined with the glucose oxidase method; the indole content, by means of HPLC; and the cAMP concentration, by ELISA. The onset of an increase in initially low intracellular cAMP content coincided with the depletion of glucose in the medium. Maximum cAMP accumulation in the cells was proportional to the concentration of initially added glucose. At the same time, the (p) ppGpp0 mutant showed a decrease in maximum cAMP levels compared to the (p)ppGpp+ parent, which was the most pronounced in the medium with 22.2 mM glucose. So, (p)ppGpp was able to positively regulate cAMP formation. The promoter of the tryptophanase operon responsible for indole biosynthesis is known to be under the positive control of catabolic repression. Therefore, in the cells of the (p)ppGpp+ strain grown in the tryptophan-free medium that were characterized by a low rate of spontaneous indole formation, its synthesis significantly increased in response to the rising cAMP level just after glucose depletion. However, this was not observed in the (p)ppGpp0 mutant cells with reduced cAMP accumulation. When tryptophan was added to the medium, both of these strains demonstrated high indole production, which was accompanied by a decrease in cAMP accumulation compared to the tryptophan-free control. Thus, under glucose depletion, (p)ppGpp can positively regulate the accumulation of both cAMP and indole, while the latter, in its turn, has a negative effect on cAMP formation.

The cyclic AMP (cAMP) phosphodiesterase CpdA required for growth, biofilm formation, motility and pathogenicity of Edwardsiella piscicida

Edwardsiella piscicida is a severe fish pathogen with wide host range, causing the huge economic losses in the aquaculture industry. Cyclic adenosine monophosphate (cAMP) as an important second messenger regulates the physiological and behavioral responses to environmental cues in eukaryotic and prokaryotic. The intracellular level of cAMP for effective activity is tightly controlled by the synthesis of adenylate cyclase, excretion and degradation of phosphodiesterase. In this study, we identified and characterized a class III cAMP phosphodiesterase, named as CpdA, in the E. piscicida. To investigate the role of CpdA in the physiology and pathogenicity, we constructed the in-frame deletion mutant of cpdA of E. piscicida, TX01ΔcpdA. The results showed that TX01ΔcpdA accumulated the higher intracellular cAMP concentration than TX01, indicating that CpdA exerted the hydrolysis of cAMP. In addition, compared to the TX01, the TX01ΔcpdA slowed growth rate, diminished biofilm formation and lost motility. More importantly, pathogenicity analysis confirmed that TX01ΔcpdA significantly impaired the ability of invading the epithelial cells, reproduction in macrophages, tissues dissemination and lethality for healthy tilapias. The most of lost properties of TX01ΔcpdA were restored partially or fully by the introduction of cpdA gene. These results suggest that cpdA is required for regulation of the physiology and virulence of E. piscicida.

Bile acids regulate SF-1 to alter cholesterol balance in adrenocortical cells via S1PR2

Glucocorticoid synthesis typically occurs in adrenal cortex and is influenced by cholesterol balance, since cholesterol is the sole precursor of steroids. Bile acids as the signaling molecules, have been shown to promote steroidogenesis in steroidogenic cells. However, whether bile acids directly regulate cholesterol balance remains elusive. In this study, we prepared cholestatic mouse models and cultured human adrenocortical cells (H295R) treated with taurochenodeoxycholic acid (TCDCA) to determine transcription levels of cholesterol metabolism associated genes and cholesterol concentrations in adrenocortical cells. Results showed that common bile duct ligation (CBDL) and chenodeoxycholic acid (CDCA) feeding elevated the mRNA levels of Abca1, Cyp51, Hmgcr, Srb1, and Mc2r in adrenals of mice. Meanwhile, the concentrations of total cholesterol and cholesteryl ester in adrenals of CBDL and CDCA-fed mice were dramatically lowered. The total and phosphorylation levels of HSL in adrenal glands of CBDL mice were also enhanced. Similarly, TCDCA treatment in H295R cells decreased intracellular concentrations of total cholesterol and cholesteryl ester and increased transcription levels of SRB1, MC2R, and HSL as well. Inhibition of bile acids’ receptor sphingosine 1‐phosphate receptor 2 (S1PR2), extracellular signal‐regulated kinase (ERK) phosphorylation, and steroidogenic factor 1 (SF-1) respectively successfully abolished effect of TCDCA on H295R cells. SF-1s was found to be phosphorylated at Thr75 in TCDCA-treated H295R cells. While a mild increase of intracellular cAMP concentration was detected upon TCDCA treatment, inhibition of PKA activity with Rp-Isomer in H295R cells failed to decrease the expression of SF-1 and its target genes. Our findings suggest that conjugated bile acids affect cholesterol balance through regulation of SF-1 in adrenocortical cells so as to provide an adequate cholesterol supply for glucocorticoid synthesis, which improves and enriches our understanding of the mechanism whereby bile acids regulate cholesterol balance to affect adrenal function.

Effect and mechanism of Zexie Decoction in promoting white adipose tissue browning/brown adipose tissue activation based on GLP-1R/cAMP/PKA/CREB pathway

This study investigated the mechanism of Zexie Decoction(ZXD) in promoting white adipose tissue browning/brown adipose tissue activation based on the GLP-1R/cAMP/PKA/CREB pathway. A hyperlipidemia model was induced by a western diet(WD) in mice, and the mice were divided into a control group, a model group(WD), and low-, medium-, and high-dose ZXD groups. An adipogenesis model was induced in 3T3-L1 cells in vitro, and with forskolin(FSK) used as a positive control, low-, medium-, and high-dose ZXD groups were set up. Immunohistochemistry and immunofluorescence results showed that compared with the WD group, ZXD promoted the expression of UCP1 in white and brown adipose tissues, and also upregulated UCP1, CPT1β, PPARα, and other genes in the cells. Western blot analysis showed a dose-dependent increase in the protein expression of PGC-1α, UCP1, and PPARα with ZXD treatment, indicating that ZXD could promote the white adipose tissue browning/brown adipose tissue activation. Hematoxylin-eosin(HE) staining results showed that after ZXD treatment, white and brown adipocytes were significantly reduced in size, and the mRNA expression of ATGL, HSL, MGL, and PLIN1 was significantly upregulated as compared with the results in the WD group. Oil red O staining and biochemical assays indicated that ZXD improved lipid accumulation and promoted lipolysis. Immunohistochemistry and immunofluorescence staining for p-CREB revealed that ZXD reversed the decreased expression of p-CREB caused by WD. In vitro intervention with ZXD increased the protein expression of CREB, p-CREB, and p-PKA substrate, and increased the mRNA level of CREB. ELISA detected an increase in intracellular cAMP concentration with ZXD treatment. Molecular docking analysis showed that multiple active components in Alismatis Rhizoma and Atractylodis Macrocephalae Rhizoma could form stable hydrogen bond interactions with GLP-1R. In conclusion, ZXD promotes white adipose tissue browning/brown adipose tissue activation both in vivo and in vitro, and its mechanism of action may be related to the GLP-1R/cAMP/PKA/CREB pathway.

Adaptive laboratory evolution of Rhodococcus rhodochrous DSM6263 for chlorophenol degradation under hypersaline condition

BackgroundNormally, a salt amount greater than 3.5% (w/v) is defined as hypersaline. Large amounts of hypersaline wastewater containing organic pollutants need to be treated before it can be discharged into the environment. The most critical aspect of the biological treatment of saline wastewater is the inhibitory/toxic effect exerted on bacterial metabolism by high salt concentrations. Although efforts have been dedicated to improving the performance through the use of salt-tolerant or halophilic bacteria, the diversities of the strains and the range of substrate spectrum remain limited, especially in chlorophenol wastewater treatment.ResultsIn this study, a salt-tolerant chlorophenol-degrading strain was generated from Rhodococcus rhodochrous DSM6263, an original aniline degrader, by adaptive laboratory evolution. The evolved strain R. rhodochrous CP-8 could tolerant 8% NaCl with 4-chlorophenol degradation capacity. The synonymous mutation in phosphodiesterase of strain CP-8 may retard the hydrolysis of cyclic adenosine monophosphate (cAMP), which is a key factor reported in the osmoregulation. The experimentally verified up-regulation of intracellular cAMP level in the evolved strain CP-8 contributes to the improvement of growth phenotype under high osmotic condition. Additionally, a point mutant of the catechol 1,2-dioxygenase, CatAN211S, was revealed to show the 1.9-fold increment on activity, which the mechanism was well explained by molecular docking analysis.ConclusionsThis study developed one chlorophenol-degrading strain with extraordinary capacity of salt tolerance, which showed great application potential in hypersaline chlorophenol wastewater treatment. The synonymous mutation in phosphodiesterase resulted in the change of intracellular cAMP concentration and then increase the osmotic tolerance in the evolved strain. The catechol 1,2-dioxygenase mutant with improved activity also facilitated chlorophenol removal since it is the key enzyme in the degradation pathway.

Polyamines modulate mouse sperm motility

Polyamines are polycationic molecules which contains two or more amino groups (-NH3 +) highly charged at physiological pH, and among them we found spermine, spermidine, putrescine, and cadaverine. They interact with proteins, nucleic acids, modulate Ca2+, K+, and Na+ channels, and protect sperm from oxidative stress. In this work, we evaluate the effect of spermine, spermidine, and putrescine on the total, progressive and kinematic parameters of motility, capacitation, acrosome reaction, also in presence and absence of the dbcAMP, an analogue of the cAMP, and the IBMX, a phosphodiesterase inhibitor. In addition, we evaluated the intracellular concentrations of cAMP [cAMP]i, and performed an in silico analysis between polyamines and the sAC from mouse to predict the possible interaction among them. Our results showed that all polyamines decrease drastically the total, progressive and the kinetic parameters of sperm motility, decrease the capacitation, and only spermidine and putrescine impeded the acquisition of acrosome reaction. Moreover, the effect of polyamines was attenuated but not countered by the addition of db-cAMP and IBMX, suggesting a possible inhibition of the sAC. Also, the presence of polyamines induced a decrease of the [cAMP]i, and the in silico analysis predicted a strong interaction among polyamines and the sAC. Overall, the evidence suggests that probably the polyamines interact and inhibit the activity of the sAC.

SAT001 Membrane Progesterone Receptors Induce Glycogenolysis In Uterine Epithelial Cells

Abstract Disclosure: M. Berg: None. M. Dean: None. In humans and cattle, 40-60% of pregnancies fail, with most losses occurring prior to or during implantation. Throughout this time, the developing embryo needs glucose to survive. The demand for glucose increases dramatically at the morula stage as the embryo enters the uterus. Yet, how glucose is regulated to match the changing needs of the embryo is unclear. Glycogen is composed of thousands of glucose molecules and is present in the uterine epithelium of many species. Our lab has shown that the glycogen content of the bovine uterine epithelium was lower during the luteal phase than at estrus. Therefore, our objective was to elucidate the role of progesterone in glycogenolysis of uterine epithelial cells. Bovine uterine epithelial (BUTE) cells were treated with insulin-like growth factor 1 (IGF1) to stimulate glycogenesis, followed by treatment for 48 hours. Progesterone decreased glycogen levels in BUTE cells by 99% (P=0.0002). RU486 did not block progesterone's effect, indicating that the effect of progesterone was not mediated by nuclear progesterone receptors (nPR). Thus, we hypothesized that the effect of progesterone is mediated by membrane progesterone receptors (mPRs). RT-PCR confirmed that BUTE cells expressed all five mPRs (α, β, γ, δ, and ε). Like progesterone, a specific mPR agonist (Org OD 02-0) decreased glycogen levels in BUTE cells by 99% (P&amp;lt;0.0001). Indicating a potential for mPRs to regulate glycogen in vivo, immunohistochemistry showed that the bovine uterine epithelium expressed high levels of mPRα. These results validate that progesterone is acting through mPRs to stimulate glycogen breakdown in BUTE cells. Once progesterone binds to the mPR, two signaling pathways can be activated, cAMP and AMPK. Neither progesterone nor Org OD 02-0 changed intracellular cAMP concentrations. In agreement, the adenylyl cyclase activator forskolin increased cAMP concentration but did not decrease glycogen. BUTE cells treated with progesterone had increased pAMPK levels at 24 hours compared to the control (P=0.0001). BUTE cells treated with an AMPK activator (D942) had a decrease in glycogen (P=0.0051). Supporting these results in vivo, pAMPK levels in the uterine epithelium were high in the bovine uterine epithelium during the luteal phase when glycogen levels were low. In a human model, progesterone and Org OD 02-0 decreased glycogen levels to a similar extent in Ishikawa cells that express mPRs but not nPRs (P=0.0027). In conclusion, progesterone acting through the mPR and AMPK to stimulate glycogenolysis may play a crucial role in providing glucose to endometrial tissue or the growing embryo. Presentation: Saturday, June 17, 2023

OR2H2 Activates CAMKKβ-AMPK-Autophagy Signaling Axis and Suppresses Senescence in VK2/E6E7 Cells.

Olfactory receptors are expressed in multiple extra-nasal tissues and these ectopic olfactory receptors mediate tissue-specific functions and regulate cellular physiology. Ectopic olfactory receptors may play key roles in tissues constantly exposed to odorants, thus the functionality of these receptors in genital tissues is of particular interest. The functionality of ectopic olfactory receptors expressed in VK2/E6E7 human vaginal epithelial cells was investigated. OR2H2 was the most highly expressed olfactory receptor expressed in VK2/E6E7 cells, and activation of OR2H2 by aldehyde 13-13, a ligand of OR2H2, increased the intracellular calcium and cAMP concentrations. Immunoblotting demonstrated that activation of OR2H2 by aldehyde 13-13 stimulated the CAMKKβ-AMPK-mTORC1-autophagy signaling axis, and that these effects were negated by OR2H2 knockdown. AMPK is known to regulate senescence; consequently, we investigated further the effect of aldehyde 13-13 on senescence. In H2O2-induced senescent cells, activation of OR2H2 by aldehyde 13-13 restored proliferation, and reduced the expression of senescence markers, P16 and P19. Additionally, aldehyde 13-13 induced apoptosis of H2O2-induced senescent cells, compared with non-senescent normal cells. In vivo, aldehyde 13-13 increased the lifespan of Caenorhabditis elegans and budding yeast. These findings demonstrate that OR2H2 is a functional receptor in VK2/E6E7 cells, and that activation of OR2H2 activates the AMPK-autophagy axis, and suppresses cellular aging and senescence, which may increase cellular health.

The RasGEF MoCdc25 regulates vegetative growth, conidiation and appressorium-mediated infection in the rice blast fungus Magnaporthe oryzae

Ras guanine nucleotide exchange factors (RasGEFs) can trigger Ras GTPase activities and play important roles in controlling various cellular processes in eukaryotes. Recently, it has been exhibited that RasGEF Cdc25 regulates morphological differentiation and pathogenicity in several plant pathogenic fungi. However, the role of RasGEFs in Magnaporthe oryzae is largely unknown. In this study, we identified and functionally characterized a RasGEF gene MoCDC25 in M. oryzae, which is orthologous to Saccharomyces cerevisiae CDC25. Targeted gene deletion mutants (ΔMocdc25) were completely nonpathogenic and were severely impaired in hyphal growth, conidiation and appressorium formation. The mutants exhibited highly sensitive response to osmotic, cell wall integrity or oxidative stresses. MoCdc25 physically interacts with the MAPK scaffold Mst50 and the putative Cdc42GEF MoScd1 in yeast two-hybrid assays. Moreover, we found that MoCdc25 was involved in regulating the phosphorylation of the MAP kinases (Pmk1, Mps1, and Osm1). In addition, the intracellular cAMP content in hyphae of the ΔMocdc25 mutants was significantly reduced compared to the parent strain Ku80 and the defect of appressorium formation of the mutants could be partially restored by the supplement of exogenous cAMP. Taken together, we conclude that the RasGEF MoCdc25 regulates vegetative growth, conidiation, appressorium formation and pathogenicity via MAPK and cAMP response pathways in M. oryzae.

Inhibitory effects of a maleimide compound on the virulence factors of Candida albicans

ABSTRACT Candidiasis caused by Candida albicans infection has long been a serious human health problem. The pathogenicity of C. albicans is mainly due to its virulence factors, which are novel targets of antifungal drugs for low risk of resistance development. In this study, we identified a maleimide compound [1-(4-methoxyphenyl)-1hydro-pyrrole-2,5-dione, MPD] that exerts effective anti-virulence activity. It could inhibit the process of adhesion, filamentation, and biofilm formation in C. albicans. In addition, it exhibited low cytotoxicity, hemolytic activity, and drug resistance development. Moreover, in Galleria mellonella-C. albicans (in vivo) infection model, the survival time of infected larvae was significantly prolonged under the treatment of MPD. Further, mechanism research revealed that MPD increased farnesol secretion by upregulating the expression of Dpp3. The increased farnesol inhibited the activity of Cdc35, which then decreased the intracellular cAMP content resulting in the inhibition of virulence factors via the Ras1-cAMP-Efg1 pathway. In all, this study evaluated the inhibitory effect of MPD on various virulence factors of C. albicans and identified the underlying mechanisms. This suggests a potential application of MPD to overcome fungal infections in clinics.

Luman regulates the activity of the LHCGR promoter

Testosterone in male mammals is mainly secreted by testicular Leydig cells, and its secretion process is regulated by the hypothalamic-pituitary-gonadal axis. After receiving the luteinizing hormone (LH) stimulus signal, the lutropin/choriogonadotropin receptor (LHCGR) on the Leydig cell membrane transfers the signal into the cell and finally increases the secretion of testosterone by upregulating the expression of steroid hormone synthase. In previous experiments, we found that interfering with the expression of the Luman protein can significantly increase testosterone secretion in MLTC-1 cells. In this experiment, we found that knockdown of Luman in MLTC-1 cells significantly increased the concentration of cAMP and upregulated the expression of AC and LHCGR. Moreover, an analysis of the activity of the LHCGR promoter by a dual luciferase reporter system showed that knockdown of Luman increased the activity of the LHCGR promoter. Therefore, we believe that knockdown of Luman increased the activity of the LHCGR promoter and upregulated the expression of LHCGR, thereby increasing the concentration of intracellular cAMP and ultimately leading to an increase of testosterone secretion by MLTC-1 cells.

Role of the adenylate cyclase/cyclic AMP pathway in oxytocin-induced lacrimal gland myoepithelial cells contraction

The aim of these studies was to investigate the involvement of the second messenger 3′,5′-cyclic adenosine monophosphate (cAMP) and its downstream effectors in oxytocin (OXT)-mediated lacrimal gland myoepithelial cell (MEC) contraction. Lacrimal gland MEC were isolated and propagated from alpha-smooth muscle actin (SMA)-GFP mice. RNA and protein samples were prepared to analyze G protein expression by RT-PCR and western blotting; respectively. Changes in intracellular cAMP concentration were measured using a competitive ELISA kit. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or a cell permeant cAMP analog, dibutyryl (db)-cAMP. In addition, inhibitors and selective agonists were used to investigate the role of cAMP effector molecules, protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using ImageJ software. The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. OXT increased intracellular cAMP in a concentration-dependent manner. FKN, IBMX and db-cAMP significantly stimulated MEC contraction. Preincubation of cells with either Myr-PKI, a specific PKA inhibitor or ESI09, an EPAC inhibitor, resulted in almost complete inhibition of both FKN- as well as OXT-stimulated MEC contraction. Finally, direct activation of PKA or EPAC using selective agonists triggered MEC contraction. We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC activation which also play a major role in OXT induced MEC contraction.

Prenylated indole-terpenoids with antidiabetic activities from Penicillium sp. HFF16 from the rhizosphere soil of Cynanchum bungei Decne.

Finding novel and effective suppression of hepatic glucagon response antidiabetic compounds is urgently required for the development of new drugs against diabetes. Fungi are well known for their ability to produce new bioactive secondary metabolites. In this study, four new prenylated indole-terpenoids (1-4), named encindolenes I-L, as well as a known analogue (5), were isolated from the fungus Penicillium sp. HFF16from the rhizosphere soil of Cynanchum bungei Decne. The structures of the compounds were elucidated by spectroscopic data and ECD analysis. In the antidiabetic activity assay, compounds 1-5 could inhibit glucagon-induced hepatic glucose output with EC50 values of 67.23, 102.1, 49.46, 25.20, and 35.96 μM, respectively, and decrease the intracellular cAMP contents in primary hepatocytes.

Platelet Phosphatidylserine Is the Critical Mediator of Increased Thrombus Formation in Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a severe adverse event that is caused by the interaction of platelets with ultra-large platelet factor 4 (PF4)/heparin IgG-immune complexes. Previous studies have shown that HIT Abs harbour the potential to interact with PLT Fc-gamma-RIIA leading to PLT activation and interplay with different actors of the innate and adaptive immune system. Despite this mechanistic insights, the role of different HIT Ab-induced PLT subpopulations that might contribute to an increased pro-thrombotic potential and severe patient outcome, remains poorly understood. In this study, we hypothesized that PF4/heparin HIT Abs induce a procoagulant PLT response that contribute to an increased pro-thrombotic environment. Using flow cytometry, we found that HIT patient Abs have the capability to induce a procoagulant (P-Selectin/phosphatidylserine double positive) PLT phenotype (p=0.0039) with increased thrombin generation potential (p<0.0001). Most importantly, the perfusion of whole blood samples that were spiked with HIT Ab-induced procoagulant PLTs resulted in significant increase of procoagulant (Annexin-V positive) PLT and fibrin deposition. Interestingly, Fc-gamma-RIIA inhibition via monoclonal antibody IV.3 resulted in a nearly complete abrogation of HIT Ab-induced thrombus formation, indicating that PLT Fc-gamma-RIIA signalling mechanisms are pivotal for Ab-induced pro-thrombotic PLT potential (p=0.0108, Figure 1A). Moreover, also the prostacyclin analogue Iloprost prevented procoagulant PLTs and increased thrombus formation despite the presence of HIT Abs and low dose (0.2 IUs/mL) heparin (p=0.0029). A finding that might be most probably due to increased intracellular concentrations of the second messenger cAMP due to increased PKA activity. To deeper investigate how increased CD62p expression and high PS externalization on the procoagulant PLT surface contribute to the prothrombotic potential of HIT Ab-induced procoagulant PLTs, we performed specific inhibition experiments. Although a marked reduction of neutrophil granulocyte deposition was observed, blocking of PLT CD62p did not reduced HIT Ab-induced increased thrombin generation or thrombus formation. In contrast, procoagulant PLTs that were treated with lactadherin, a PS binding and blocking protein resulted in a significant reduction of thrombin generation (p=0.0040) and thrombus formation compared to the vehicle control (p=0.0005, Figure 1B). Findings of our study indicate that HIT patient Abs harbour the ability to induce procoagulant PLTs via PLT FcγRIIA mediated signalling pathways. The observation that Iloprost reduced the formation of procoagulant PLTs and most importantly inhibits thrombus formation indicates a potential therapeutic use. Most importantly, based on our mechanistic studies we conclude that increased PS on the surface of procoagulant PLTs might be the causing factor for increased pro-thrombotic potential observed in HIT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Double broncholytic combination of umeclidnium/ vilanterol in COPD therapy: issues of efficacy and safety

Inhaled bronchodilators are the basis of pharmacological therapy of chronic obstructive pulmonary disease. Bronchodilation can be reached in two different ways: by stimulation of β2 -adrenoreceptors and inhibition of M3-cholinoreceptors. Combined long acting bronchodilators show additional effect on lung function and clinical outcomes of the disease. This article covers the issues of efficacy and safety of different combined bronchodilators. Chronic obstructive pulmonary disease (COPD) is a disease characterized by the limitation of air flow velocity, which is reversible incompletely. The restriction of air flow velocity is usually progressive and is associated with inflammatory response of the lungs to pathogenic particles or gases, with tobacco smoking being the main cause. COPD leads to a number of significant systemic effects that can worsen the course of the disease. It is important that COPD can be prevented and treated. The Global Strategy for the Diagnosis, Treatment and Prevention of Chronic Obstructive Pulmonary Disease (GOLD) considers long-acting inhaled bronchodilators as the basis for long-term maintenance therapy of chronic obstructive pulmonary disease. GOLD recommends the use of both long-acting anticholinergic drugs (LAMA) and long-acting β2-agonists (LABA) in all categories of patients, including as the first line in people with severe symptoms and low risk of exacerbations. bronchodilators reduce the severity of shortness of breath and other symptoms of the disease, and also increase exercise tolerance. The individual components have different application points: LAMA inhibit the effect of acetylcholine mainly on M1- and M3-muscarinic receptors, LABA stimulate β2-adrenergic receptors, increasing the content of intracellular cAMP and causing bronchodilation. Both classes of drugs potentiate each other’s activity, increasing the relaxing effect on the smooth muscles of the bronchi.