Elaidic acid induced NLRP3 inflammasome activation via ERS-MAPK signaling pathways in Kupffer cells.

Trans fatty acids (TFA) in food can cause liver inflammation. Activation of NOD-like receptor protein-3 (NLRP3) inflammasome is a key factor in the regulation of inflammation. Accumulating evidence suggests that ERS-induced NLRP3 inflammasome activation underlies the pathological basis of various inflammatory diseases, but the precise mechanism has not been fully elucidated. Therefore, this paper focused on TFA, represented by elaidic acid (EA), to investigate the mechanism of liver inflammation. Levels of mRNA and protein were detected by RT-qPCR and Western blotting, the release of proinflammatory cytokines was measured by ELISA, and intracellular Ca2+ levels were determined by flow cytometer using Fluo 4-AM fluorescent probes. Our research indicated that EA induced the endoplasmic reticulum stress (ERS) response in Kupffer cells (KCs), accompanied by the activation of the mitogen-activated protein kinase (MAPK) signaling pathway, which resulted in NLRP3 inflammasome formation, and eventually increased the release of inflammatory factors. NLRP3 inflammasome activation was inhibited when KCs were pretreated with ERS inhibitors (4-PBA) and MAPK selective inhibitors. Furthermore, when ERS was blocked, the MAPK pathway was inhibited.

Bioisosteric Modification of To042: Synthesis and Evaluation of Promising Use-Dependent Inhibitors of Voltage-Gated Sodium Channels.

Three analogues of To042, a tocainide-related lead compound recently reported for the treatment of myotonia, were synthesized and evaluated in vitro as skeletal muscle sodium channel blockers possibly endowed with enhanced use-dependent behavior. Patch-clamp experiments on hNav1.4 expressed in HEK293 cells showed that N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine, the aryloxyalkyl bioisostere of To042, exerted a higher use-dependent block than To042 thus being able to preferentially block the channels in over-excited membranes while preserving healthy tissue function. It also showed the lowest active transport across BBB according to the results of P-glycoprotein (P-gp) interacting activity evaluation and the highest cytoprotective effect on HeLa cells. Quantum mechanical calculations and dockings gave insights on the most probable conformation of the aryloxyalkyl bioisostere of To042 in solution and the target residues involved in the binding, respectively. Both approaches indicated the conformations that might be adopted in both the unbound and bound state of the ligand. Overall, N-[(naphthalen-1-yl)methyl]-4-[(2,6-dimethyl)phenoxy]butan-2-amine exhibits an interesting toxico-pharmacological profile and deserves further investigation.

AMPA induced cognitive impairment in rats: Establishing the role of endoplasmic reticulum stress inhibitor, 4-PBA.

Glutamate excitotoxicity and endoplasmic reticulum (ER) recently have been found to be instrumental in the pathogenesis of various neurodegenerative diseases. However, the paucity of literature deciphering the inter-linkage among glutamate receptors, behavioral alterations, and ER demands thorough exploration. Reckoning the aforesaid concerns, a prospective study was outlined to delineate the influence of ER stress inhibition via 4-phenylbutyric acid (PBA) on α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) excitotoxicity-induced behavioral aspects and possible ER stress-glutamate linkage. Male SD rats were randomly divided into four groups namely sham (surgical control+vehicle, group 1), AMPA-induced excitotoxic group 2 receive a single intra-hippocampal injection of 10 mM AMPA, group 3 received AMPA along with PBA (i.p, 100 mg/kg body weight) for 15 days, and group 4 received PBA alone. Behavioral analyses were performed prior to the sacrifice of animals and hippocampus was extracted thereafter for further analysis. AMPA-induced excitotoxicity exhibited significant impairment of locomotion as well as cognitive functions. The levels of neurotransmitters such as dopamine, homo vanillic acid (HVA), norepinephrine, and serotonin were reduced accompanied by reduced expression of GLUR1 and GLUR4 (glutamate receptor) as well as loss of neurons in different layers of hippocampus. ER stress markers were upregulated upon AMPA excitotoxicity. However, chemical chaperone PBA supplementation remarkably mitigated the behavioral alterations along with expression of glutamate and ER stress intermediates/markers in AMPA excitotoxic animals. Therefore, the present exploration convincingly emphasizes the significance of ER stress and its inhibition via PBA in combating cognitive impairment as well as improving locomotion in excitotoxic animals.

Suppression of Endoplasmic Reticulum Stress by 4-PBA Protects Against Hyperoxia-Induced Acute Lung Injury via Up-Regulating Claudin-4 Expression.

Endoplasmic reticulum (ER) stress that disrupts ER function can occur in response to a wide variety of cellular stress factors leads to the accumulation of unfolded and misfolded proteins in the ER. Many studies have shown that ER stress amplified inflammatory reactions and was involved in various inflammatory diseases. However, little is known regarding the role of ER stress in hyperoxia-induced acute lung injury (HALI). This study investigated the influence of ER stress inhibitor, 4-phenyl butyric acid (4-PBA), in mice with HALI. Treatment with 4-PBA in the hyperoxia groups significantly prolonged the survival, decreased lung edema, and reduced the levels of inflammatory mediators, lactate dehydrogenase, and protein in bronchoalveolar lavage fluid, and increased claudin-4 protein expression in lung tissue. Moreover, 4-PBA reduced the ER stress-related protein expression, NF-κB activation, and apoptosis in the lung tissue. In in vitro study, 4-PBA also exerted a similar effect in hyperoxia-exposed mouse lung epithelial cells (MLE-12). However, when claudin-4 siRNA was administrated in mice and MLE-12 cells, the protective effect of 4-PBA was abrogated. These results suggested that 4-PBA protected against hyperoxia-induced ALI via enhancing claudin-4 expression.

Biased action of the CXCR4-targeting drug plerixafor is essential for its superior hematopoietic stem cell mobilization.

Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.

The Role of Unfolded Protein Response in Human Intervertebral Disc Degeneration: Perk and IRE1-α as Two Potential Therapeutic Targets.

Inflammation plays a key role in intervertebral disc degeneration (IDD). The association between inflammation and endoplasmic reticulum (ER) stress has been observed in many diseases. However, whether ER stress plays an important role in IDD remains unclear. Therefore, this study is aimed at investigating the expression of ER stress in IDD and at exploring the underlying mechanisms of IDD, ER stress, and inflammation. The expression of ER stress was activated in nucleus pulposus cells from patients who had IDD (D-NPCs) compared with patients without IDD (N-NPCs); and both the proliferation and synthesis capacity were decreased by inducer tunicamycin (Tm) and proinflammatory cytokines. Pretreatment of NPCs with 4-phenyl butyric acid (4-PBA) prevented the inflammatory cytokine-induced upregulation of unfolded protein response- (UPR-) related proteins and recovered cell synthetic ability. Furthermore, proinflammatory cytokine treatment significantly upregulated the expression of inositol-requiring protein 1 (IRE1-α) and protein kinase RNA-like ER kinase (PERK), but not activating transcription factor 6 (ATF6). Finally, knockdown of IRE1-α and PERK also restored the biological activity of NPCs. Our findings identified that IRE1-α and PERK might be the potential targets for IDD treatment, which may help illustrate the underlying mechanism of ER stress in IDD.

WTAP promotes myocardial ischemia/reperfusion injury by increasing endoplasmic reticulum stress via regulating m6A modification of ATF4 mRNA.

Myocardial infarction (MI) is one of the leading causes of death. Wilms' tumor 1-associating protein (WTAP), one of the components of the m6A methyltransferase complex, has been shown to affect gene expression via regulating mRNA modification. Although WTAP has been implicated in various diseases, its role in MI is unclear. In this study, we found that hypoxia/reoxygenation (H/R) time-dependently increased WTAP expression, which in turn promoted endoplasmic reticulum (ER) stress and apoptosis, in human cardiomyocytes (AC16). H/R effects on ER stress and apoptosis were all blocked by silencing of WTAP, promoted by WTAP overexpression, and ameliorated by administration of ER stress inhibitor, 4-PBA. We then investigated the underlying molecular mechanism and found that WTAP affected m6A methylation of ATF4 mRNA to regulate its expression, and that the inhibitory effects of WTAP on ER stress and apoptosis were ATF4 dependent. Finally, WTAP's effects on myocardial I/R injury were confirmed in vivo. WTAP promoted myocardial I/R injury through promoting ER stress and cell apoptosis by regulating m6A modification of ATF4 mRNA. These findings highlight the importance of WTAP in I/R injury and provide new insights into therapeutic strategies for MI.

Mycoplasmas bovis P48 induces apoptosis in EBL cells via an endoplasmic reticulum stress-dependent signaling pathway.

Mycoplasma bovis (M. bovis) is a small bacterium that lacks a cell wall. M. bovis infection can result in chronic pneumonia and polyarthritis syndrome (CPPS), otitis media, conjunctivitis, and meningitis in feedlot cattle and mastitis in dairy cattle. To gain more understanding of the mechanism of M. bovis and host interaction, this study focused on P48, an important membrane protein involved in M. bovis adhesion, proliferation and virulence. In this study, exogenous P48 protein was introduced to explore its function in embryonic bovine lung (EBL) cells by recombinant vector and protein purification. We found that M. bovis infection inhibited EBL cells growth and enhanced apoptosis. Both intracellular and extracellular P48 protein treatment also induce apoptosis. Moreover, P48 activates endoplasmic reticulum (ER) stress response via increasing ER stress markers expression. To further explore the underlying mechanism, we performed inhibition experiments using ER stress inhibitor 4-PBA and specific siRNA interference against GRP78, and found that P48 protein modulated EBL cells apoptosis in an ER stress signaling-dependent manner. This study provided more data to further understand M. bovis infection mechanism and develop effective anti-mycoplasma strategy.

Amphetamine-like Neurochemical and Cardiovascular Effects of α-Ethylphenethylamine Analogs Found in Dietary Supplements.

Dietary supplements often contain additives not listed on the label, including α-ethyl homologs of amphetamine such as N,α-diethylphenethylamine (DEPEA). Here, we examined the neurochemical and cardiovascular effects of α-ethylphenethylamine (AEPEA), N-methyl-α-ethylphenethylamine (MEPEA), and DEPEA as compared with the effects of amphetamine. All drugs were tested in vitro using uptake inhibition and release assays for monoamine transporters. As expected, amphetamine acted as a potent and efficacious releasing agent at dopamine transporters (DAT) and norepinephrine transporters (NET) in vitro. AEPEA and MEPEA were also releasers at catecholamine transporters, with greater potency at NET than DAT. DEPEA displayed fully efficacious release at NET but weak partial release at DAT (i.e., 40% of maximal effect). In freely moving, conscious male rats fitted with biotelemetry transmitters for physiologic monitoring, amphetamine (0.1-3.0 mg/kg, s.c.) produced robust dose-related increases in blood pressure (BP), heart rate (HR), and motor activity. AEPEA (1-10 mg/kg, s.c.) produced significant increases in BP but not HR or activity, whereas DEPEA and MEPEA (1-10 mg/kg, s.c.) increased BP, HR, and activity. In general, the phenethylamine analogs were approximately 10-fold less potent than amphetamine. Our results show that α-ethylphenethylamine analogs are biologically active. Although less potent than amphetamine, they produce cardiovascular effects that could pose risks to humans. Given that MEPEA and DEPEA increased locomotor activity, these substances may also have significant abuse potential. SIGNIFICANCE STATEMENT: The α-ethyl homologs of amphetamine have significant cardiovascular, behavioral, and neurochemical effects in rats. Given that these compounds are often not listed on the ingredient labels of dietary supplements, these compounds could pose a risk to humans using these products.

A novel dopamine D3R agonist SK609 with norepinephrine transporter inhibition promotes improvement in cognitive task performance in rodent and non-human primate models of Parkinson's disease.

Mild cognitive impairment is present in a number of neurodegenerative disorders including Parkinson's disease (PD). Mild cognitive impairment in PD (PD-MCI) often manifests as deficits in executive functioning, attention, and spatial and working memory. Clinical studies have suggested that the development of mild cognitive impairment may be an early symptom of PD and may even precede the onset of motor impairment by several years. Dysfunction in several neurotransmitter systems, including dopamine (DA), norepinephrine (NE), may be involved in PD-MCI, making it difficult to treat pharmacologically. In addition, many agents used to treat motor impairment in PD may exacerbate cognitive impairment. Thus, there is a significant unmet need to develop therapeutics that can treat both motor and cognitive impairments in PD. We have recently developed SK609, a selective, G-protein biased signaling agonist of dopamine D3 receptors. SK609 was successfully used to treat motor impairment and reduce levodopa-induced dyskinesia in a rodent model of PD. Further characterization of SK609 suggested that it is a selective norepinephrine transporter (NET) inhibitor with the ability to increase both DA and NE levels in the prefrontal cortex. Pharmacokinetic analysis of SK609 under systemic administration demonstrated 98% oral bioavailability and high brain distribution in striatum, hippocampus and prefrontal cortex. To evaluate the effects of SK609 on cognitive deficits of potential relevance to PD-MCI, we used unilateral 6-hydroxydopamine (6-OHDA) lesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated cynomolgus macaques, with deficits in performance in a sustained attention and an object retrieval task, respectively. SK609 dose dependently improved the performance of 6-OHDA-lesioned rats, with peak performance achieved using a 4 mg/kg dose. This improvement was predominantly due to a significant reduction in the number of misses and false alarm errors, contributing to an increase in sustained attention. In MPTP-lesioned monkeys, this same dose also improved performance in an object retrieval task, significantly reducing cognitive errors (barrier reaches) and motor errors (fine motor dexterity problems). These data demonstrate that SK609 with its unique pharmacological effects on modulating both DA and NE can ameliorate cognitive impairment in PD models and may provide a therapeutic option to treat both motor and cognitive impairment in PD patients.

Interaction of Estradiol and Endoplasmic Reticulum Stress in the Development of Esophageal Carcinoma.

Gender differences in esophageal cancer patients indicate that estradiol may have antitumor effects on esophageal cancer. The initiation of endoplasmic reticulum stress (ERS) can induce apoptosis in esophageal cancer cells. However, it is still unknown whether estradiol inhibits the development of esophageal cancer by activating ERS pathway. In this study, the gender difference in the development of esophageal cancer was observed by analyzing clinical data and the experimental tumor xenografts in mice. Meanwhile, we investigated the mechanism of ERS in estradiol-mediated inhibition of esophageal cancer using esophageal squamous cell carcinoma cell line EC109. The proportion of male patients with esophageal cancer was significantly higher than female patients. Meanwhile, male patients were prone to have adventitial invasion. The weight of transplanted tumors in female mice was significantly smaller than that in male mice. In vitro experiments showed estradiol inhibits the viability and migration of EC109 cells by increasing the expression of ERS-related proteins, whereas ERS inhibitor 4-PBA abolished the effects of estradiol. In conclusion, our data demonstrate that sex difference exists in the occurrence of esophageal cancer. Estradiol can inhibit the viability and migration of esophageal cancer cells through the activation of ERS, providing a novel insight for esophageal cancer development, treatment, and prevention.

High T3 Induces ß-Cell Insulin Resistance via Endoplasmic Reticulum Stress.

Hyperthyroidism can cause glucose metabolism disorders and insulin resistance. Insulin resistance in muscle and adipose tissues has been extensively studied, whereas investigations on ß-cell insulin resistance are limited. This study preliminarily explored the effects of high T3 levels on ß-cell line (MIN6) insulin resistance, as well as the roles of endoplasmic reticulum stress (ERS). In this study, we treated ß-cell line with T3, with or without an inhibitor of phosphotyrosine phosphatases (PTPs, sodium vanadate) or ERS inhibitor (4-PBA). The results indicated that high levels of T3 significantly inhibited insulin secretion in ß-cell line. In addition, we observed an upregulation of p-IRS-1ser307 and downregulation of Akt. These results can be corrected by sodium vanadate. Moreover, high T3 levels upregulate the ERS-related proteins PERK, IRE1, ATF6, and GRP78, as well as ERS-related apoptosis CHOP and caspase-12. Similarly, this change can be corrected by 4-PBA. These results suggest that high T3 levels can induce insulin resistance in ß-cell line by activating ERS and the apoptotic pathway.

Butaphosphan Effects on Glucose Metabolism Involve Insulin Signaling and Depends on Nutritional Plan.

Butaphosphan is an organic phosphorus compound used in several species for the prevention of rapid catabolic states, however, the mechanism of action remains unclear. This study aimed at determining the effects of butaphosphan on energy metabolism of mice receiving a normal or hypercaloric diet (HCD) and submitted or not to food restriction. Two experiments were conducted: (1) during nine weeks, animals were fed with HCD (n = 28) ad libitum, and at the 10th week, were submitted to food restriction and received butaphosphan (n = 14) or saline injections (n = 14) (twice a day, for seven days) and; (2) during nine weeks, animals were fed with a control diet (n = 14) or HCD (n = 14) ad libitum, and at the 10th week, all animals were submitted to food restriction and received butaphosphan or saline injections (twice a day, for seven days). In food restriction, butaphosphan preserved epididymal white adipose tissue (WAT) mass, increased glucose, NEFA, and the HOMA index. In mice fed HCD and submitted to food restriction, the butaphosphan preserved epididymal WAT mass. Control diet influences on PI3K, GCK, and Irs1 mRNA expression. In conclusion, butaphosphan increased blood glucose and reduced fat mobilization in overweight mice submitted to caloric restriction, and these effects are influenced by diet.

Efficacy evaluation and mechanism study on inhibition of breast cancer cell growth by multimodal targeted nanobubbles carrying AMD070 and ICG.

To construct targeted nanobubbles carrying both small-molecule CXCR4 antagonist AMD070 and light-absorbing material indocyanine green (ICG), and to study their in vitro multimodal imaging, as well as their mechanism and efficacy of inhibition of breast cancer cell growth. Nanobubbles carrying AMD070 and ICG (ICG-TNBs) were constructed by carbodiimide reaction and mechanical oscillation. The physical characteristics and in vitro multimodal imaging were determined. The binding potential of ICG-TNBs to human breast cancer cells were observed by laser confocal microscopy. CCK-8 and flow cytometry were used to analyze the role of ICG-TNBs + US in inhibiting proliferation and inducing apoptosis of tumor cells. Flow cytometry and Western blotting are used to analyse the ROS generation and molecular mechanisms. ICG-TNBs had a particle size of 497.0 ± 29.2 nm and a Zeta potential of -8.05 ± 0.73 mV. In vitro multimodal imaging showed that the image signal intensity of ICG-TNBs increased with concentration. Targeted binding assay confirmed that ICG-TNBs could specifically bind to MCF-7 cells (CXCR4 positive), but not to MDA-MB-468 cells (CXCR4 negative). CCK-8 assay and flow cytometry analysis showed that ICG-TNBs + US could significantly inhibit the growth of MCF-7 breast cancer cells and promote their apoptosis. Flow cytometry and Western blotting showed that ICG-TNBs + US could significantly raise generation of ROS, reduce the expression of CXCR4, inhibit phosphorylation of Akt, and increase the expression of Caspase3 and Cleaved-caspase3. This indicated that ICG-TNBs could effectively inhibit and block the SDF-1/CXCR4 pathway, thus leading to the apoptosis of MCF-7 cells. ICG-TNBs can specifically bind to CXCR4 positive breast cancer cells, furthermore inhibit growth and promote apoptosis of breast cancer cells combined with ultrasonic irradiation by blocking the SDF-1/CXCR4 pathway. This study introduces a novel concept, method and mechanism for integration of targeted diagnosis and treatment of breast cancer.

Selenium Attenuates Chronic Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in Mouse Granulosa Cells.

Heat stress induces apoptosis in various cells. Selenium, an essential micronutrient, has beneficial effects in maintaining the cellular physiological functions. However, its potential protective action against chronic heat stress (CHS)-induced apoptosis in granulosa cells and the related molecular mechanisms are not fully elucidated. In this study, we investigated the roles of selenium in CHS-induced apoptosis in mouse granulosa cells and explored its underlying mechanism. The heat treatment for 6-48 h induced apoptosis, potentiated caspase 3 activity, increased the expression levels of apoptosis-related gene BAX and ER stress markers, glucose-regulated protein 78 (GRP78), and CCAAT/enhancer binding protein homologous protein (CHOP) in mouse granulosa cells. The treatment with ER stress inhibitor 4-PBA significantly attenuated the adverse effects caused by CHS. Selenium treatment significantly attenuated the CHS- or thapsigargin (Tg, an ER stress activator)-induced apoptosis, potentiation of caspase 3 activity, and the increased protein expression levels of BAX, GRP78, and CHOP. Additionally, treatment of the cells with 5 ng/mL selenium significantly ameliorated the levels of estradiol, which were decreased in response to heat exposure. Consistently, administering selenium supplement alleviated the hyperthermia-caused reduction in the serum estradiol levels in vivo. Together, our findings indicate that selenium has protective effects on CHS-induced apoptosis via inhibition of the ER stress pathway. The current study provides new insights in understanding the role of selenium during the process of heat-induced cell apoptosis.

Effects of Stearic Acid on Proliferation, Differentiation, Apoptosis, and Autophagy in Porcine Intestinal Epithelial Cells.

BACKGROUND: Stearic acid (SA), a saturated long-chain fatty acid consisting of 18 carbon atoms, is widely found in feed ingredients, such as corn, soybeans, and wheat. However, the roles of SA in the renewal of intestinal epithelial cells remain unclear. METHODS AND RESULTS: In the present study, we found that 0.01-0.1 mM SA promoted IPEC-J2 cell differentiation and did not affect IPEC-J2 cell viability. In addition, the results showed that the viability of IPEC-J2 cells was inhibited by SA in a time- and dose-dependent manner at high concentrations. Flow cytometry and western blot analysis suggested that SA induced apoptosis, autophagy and ER stress in cells. In addition, the amounts of triglyceride were significantly increased upon challenge with SA. Moreover, the decrease in the viability of cells induced by SA could be attenuated by 4-PBA, an inhibitor of ER stress. CONCLUSION: In summary, SA accelerated IPEC-J2 cell differentiation at 0.01-0.1 mM. Furthermore, SA induced IPEC-J2 cell apoptosis and autophagy by causing ER stress.

Pharmacokinetics of N-ethylpentylone and its effect on increasing levels of dopamine and serotonin in the nucleus accumbens of conscious rats.

N-Ethylpentylone (NEP) is one of the most confiscated synthetic cathinones in the world. However, its pharmacology and pharmacokinetics remain largely unknown. In this study, the pharmacokentics of NEP in rat nucleus accumbens (NAc) was assessed via brain microdialysis after the intraperitoneal (ip) administration of NEP (20 or 50 mg/kg). The concentrations of dopamine (DA) and serotonin (5-HT) and their metabolites, including 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and 5-hydroxyindoleacetic acid (5-HIAA), were simultaneously monitored to elucidate the pharmacological effect of NEP. In addition, the plasma levels of NEP were also assessed. The pharmacokinetics of NEP showed a dose-related pattern, with NEP rapidly passing through the blood-brain barrier and reaching a maximum concentration (Cmax ) at approximately 40-minutes postdose. Approximately 4% of plasma NEP was distributed to the NAc, and considering a homogeneous brain distribution, over 90% of plasma NEP was potentially distributed to the brain. High values of area under curve (AUC) and mean residence time (MRT) of NEP were observed in both the NAc and plasma, indicating large and long-lasting effects. NEP elicited dose-related increases in microdialysate DA and 5-HT and increased the concentration of 3-MT in a dose-related manner. However, the rate of DA converted into 3-MT was unaffected. NEP had a negative effect on the rates of which DA and 5-HT were transformed into DOPAC and 5-HIAA, respectively. In summary, NEP rapidly entered the NAc and showed a long-lasting effect. In addition, DA increased more significantly than 5-HT, indicating a large potential for NEP abuse.

Epigallocatechin-3-gallate (EGCG) inhibits aggregation of pulmonary fibrosis associated mutant surfactant protein A2 via a proteasomal degradation pathway.

BACKGROUND/AIMS: Epigallocatechin-3-gallate (EGCG), a major catechin found in green tea, plays an important anti-tumor role and is involved in various other biological processes, such as, neuroprotection by prevention of aggregation of misfolded proteins generated because of genetic defects. Surfactant protein A2 mutations (G231V and F198S) have been identified to be associated with pulmonary fibrosis and lung cancer, and these mutations cause protein aggregation, instability as well as secretion deficiency. The present study focused on investigating the inhibitory effects of EGCG on aggregation of mutant SP-A2 and elucidating the potential mechanisms underlying this action. METHODS: Wild-type and mutant SP-A2 were transiently expressed in CHO-K1 cells. The aggregated and soluble proteins were separated into NP-40-insoluble and NP-40-soluble fractions. Protein stability was validated by chymotrypsin limited proteolysis assay. Western blot and RT-PCR were used to determine the protein and mRNA expression level, respectively. RESULTS: Mutant SP-A2 alone or wild-type SP-A2 co-expressed with G231V formed NP-40-insoluble aggregates in CHO-K1 cells. EGCG significantly suppressed this aggregation and alleviated mutant SP-A2 accumulation in the ER. When combined with 4-PBA, EGCG treatment completely blocked mutant SP-A2 aggregate formation. Though secretion of mutant protein was not affected, EGCG facilitated protein instability in both wild-type and mutant protein. Importantly, MG132, a proteasome inhibitor, reversed EGCG-induced aggregate reduction. CONCLUSIONS: EGCG inhibits aggregation of misfolded SP-A2 via induction of protein instability and activation of proteasomal pathway for aggregate degradation.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development.

Around 60-80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus-oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.