Levels Of Subunits Research Articles

Improving the solubility of wheat gluten: Establishing and characterizing wheat gluten-phosvitin complex at molecular and subunit levels

Wheat gluten (WG) has excellent processing properties, however, its utilization in food industry has been largely restricted for the low solubility. We are interested in improving its solubility with simple and effective approach to broaden its application. Phosvitin (PSV) is a kind of yolk proteins with high solubility. In this work, a co-soluble system (WG-PSV complex) has been established under pH-shifting treatment. After that, the solubility of WG is effectively increased from 16.2% to 43.1%. The WG-PSV complex has smaller particles, a higher Zeta-potential, and a more flexible secondary structure compared to native WG. Meanwhile, the free sulfhydryl group content in WG-PSV complex was 12.24 μmol/g, which was higher than that of native WG (9.05 μmol/g), and its free amino group was much lower than that of native WG. In addition, the proteomics was used to compare the subunit composition in WG-PSV complex and native WG to identify key subunits related to improvement of solubility. It shows that compared to native WG, the subunits A0A3B5ZSZ8 and A0A1D5UUP9 derived from WG have been significantly upregulated, and Q306F8, A0A0E3UQV1 and Q8H738 have been significantly downregulated. In conclusion, pH-shifting is a successful method to establish WG-PSV complex to effectively improve the solubility of WG. And disulfide bonds, amide bonds and hydrophobic contacts were the main interaction forces. Some key subunits were identified to largely determine the forming of protein complex. This study provides a theoretical foundation for enhancing solubility of WG, which would widely expand its applications in food industry.

Polyscias scutellaria Aqueous Leaves Extract Increases Insulin Levels and Improves Mammary Gland Histology in Lactating Rats

BACKGROUND: Exclusive breastfeeding could be a protective step to avoid infant stunting. During lactation, several hormones are involved in milk secretion, including insulin. Polyscias scutellaria is a perennial plant which has traditionally been used to increase breast milk production. This study was conducted to examine the impact of P. scutellaria leaf extract on the mammary gland histology, expression of insulin in the mammary gland, as well as plasma insulin and the insulin receptor levels.METHODS: Five female unmated rats (UR) and twenty female lactating rats were divided into five groups, namely UR, lactating rats only (LRO), lactating rats treated with Asifit (LRA), lactating rats treated with PSAE at a dosage of 250 and 500 mg/kg body weight (BW) (LRPSAE 250 and 500). Treatments were given orally for 14 days. The dams were sacrificed after the weaning stage (third week after parturition), and the serum, mammary gland, liver, and muscle were collected for further analysis.RESULTS: The histoarchitecture of mammary gland between the LRA and LRPSAE groups were similar. The LRPSAE 250 group had higher plasma level and immunofluorescent expression of insulin than the LRA group. PSAE did not affect insulin receptor beta subunit (INSR-β) levels in both liver and muscle of lactating rats.CONCLUSION: PSAE could be used as an herbal treatment to increase breast milk production by improving mammary gland histology and maintaining the mother's insulin levels.KEYWORDS: Polyscias scutellaria, lactating rats, insulin, INSR-β

Inhibiting NLRP3 inflammasome signaling pathway promotes neurological recovery following hypoxic-ischemic brain damage by increasing p97-mediated surface GluA1-containing AMPA receptors

BackgroundThe nucleotide-binding oligomeric domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is believed to be a key mediator of neuroinflammation and subsequent secondary brain injury induced by ischemic stroke. However, the role and underlying mechanism of the NLRP3 inflammasome in neonates with hypoxic-ischemic encephalopathy (HIE) are still unclear.MethodsThe protein expressions of the NLRP3 inflammasome including NLRP3, cysteinyl aspartate specific proteinase-1 (caspase-1) and interleukin-1β (IL-1β), the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionicacid receptor (AMPAR) subunit, and the ATPase valosin-containing protein (VCP/p97), were determined by Western blotting. The interaction between p97 and AMPA glutamate receptor 1 (GluA1) was determined by co-immunoprecipitation. The histopathological level of hypoxic-ischemic brain damage (HIBD) was determined by triphenyltetrazolium chloride (TTC) staining. Polymerase chain reaction (PCR) and Western blotting were used to confirm the genotype of the knockout mice. Motor functions, including myodynamia and coordination, were evaluated by using grasping and rotarod tests. Hippocampus-dependent spatial cognitive function was measured by using the Morris-water maze (MWM).ResultsWe reported that the NLRP3 inflammasome signaling pathway, such as NLRP3, caspase-1 and IL-1β, was activated in rats with HIBD and oxygen-glucose deprivation (OGD)-treated cultured primary neurons. Further studies showed that the protein level of the AMPAR GluA1 subunit on the hippocampal postsynaptic membrane was significantly decreased in rats with HIBD, and it could be restored to control levels after treatment with the specific caspase-1 inhibitor AC-YVAD-CMK. Similarly, in vitro studies showed that OGD reduced GluA1 protein levels on the plasma membrane in cultured primary neurons, whereas AC-YVAD-CMK treatment restored this reduction. Importantly, we showed that OGD treatment obviously enhanced the interaction between p97 and GluA1, while AC-YVAD-CMK treatment promoted the dissociation of p97 from the GluA1 complex and consequently facilitated the localization of GluA1 on the plasma membrane of cultured primary neurons. Finally, we reported that the deficits in motor function, learning and memory in animals with HIBD, were ameliorated by pharmacological intervention or genetic ablation of caspase-1.ConclusionInhibiting the NLRP3 inflammasome signaling pathway promotes neurological recovery in animals with HIBD by increasing p97-mediated surface GluA1 expression, thereby providing new insight into HIE therapy.

X- and y-type thioredoxins maintain redox homeostasis on photosystem I acceptor side under fluctuating light.

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.

Mass spectrometry characterization of antibodies at the intact and subunit levels: From targeted to large-scale analysis

Antibodies are one of the most formidable molecular weapons available to our immune system. Their high specificity against a target (antigen) and capability of triggering different immune responses (e.g., complement system activation and antibody-dependent cell-mediated cytotoxicity) make them ideal drugs to fight many different human diseases. Currently, both monoclonal antibodies and more complex molecules based on the antibody scaffold are used as biologics. Naturally, such highly heterogeneous molecules require dedicated analytical methodologies for their accurate characterization. Mass spectrometry (MS) can define the presence and relative abundance of multiple features of antibodies, including critical quality attributes. The combination of small and large variations within a single molecule can only be determined by analyzing intact antibodies or their large (25–100 kDa) subunits. Hence, top-down (TD) and middle-down (MD) MS approaches have gained popularity over the last decade. In this Young Scientist Feature we discuss the evolution of TD and MD MS analysis of antibodies, including the new frontiers that go beyond biopharma applications. We will show how this field is now moving from the “quality control” analysis of a known, single antibody to the high-throughput investigation of complex antibody repertoires isolated from clinical samples, where the ultimate goal is represented by the complete gas-phase sequencing of antibody molecules without the need of any a priori knowledge.

Involvement of NO in V-ATPase Regulation in Cucumber Roots under Control and Cadmium Stress Conditions.

Nitric oxide (NO) is a signaling molecule that participates in plant adaptation to adverse environmental factors. This study aimed to clarify the role of NO in the regulation of vacuolar H+-ATPase (V-ATPase) in the roots of cucumber seedlings grown under control and Cd stress conditions. In addition, the relationship between NO and salicylic acid (SA), as well as their interrelations with hydrogen sulfide (H2S) and hydrogen peroxide (H2O2), have been verified. The effect of NO on V-ATPase was studied by analyzing two enzyme activities, the expression level of selected VHA genes and the protein level of selected VHA subunits in plants treated with a NO donor (sodium nitroprusside, SNP) and NO biosynthesis inhibitors (tungstate, WO42- and N-nitro-L-arginine methyl ester, L-NAME). Our results indicate that NO functions as a positive regulator of V-ATPase and that this regulation depends on NO generated by nitrate reductase and NOS-like activity. It was found that the mechanism of NO action is not related to changes in the gene expression or protein level of the V-ATPase subunits. The results suggest that in cucumber roots, NO signaling interacts with the SA pathway and, to a lesser extent, with two other known V-ATPase regulators, H2O2 and H2S.

Monosodium Glutamate Effect on The Expression of ɑ7nACHR and ɑ4nACHR Subunits in The Testicular Tissue.

Monosodium glutamate (MSG) is a popular food flavor enhancer, and a glutamate subset that induces different toxicities such as hepatotoxicity, neurotoxicity, reproductive toxicity, and nephrotoxicity. This study was conducted to assess the effects of MSG on the α7 and α4 nicotinic acetylcholine receptor (nACHR) protein subunits expression of adult rat testis and the safety role of vitamin C. For this experimental research, 24 rats were haphazardly grouped into four equal groups (n=6) and orally gavaged for 30 days as follows: control group (distilled water gavage), MSG group (3 g/kg/b.w/ day), vitamin C group (150 mg/kg/b.w/day), and MSG+vitamin C group (3 g/kg/b.w/day+150 mg/kg/b.w/day, respectively) that rats of all groups on the 30th day were anesthetized, and the left testes were used for of α4 and α7 nACHR protein subunit evaluation by immunohistochemistry (IHC). Statistical computations were performed using Graph Pad Prism software. The present study revealed a significant reduction in the expression and optical density (OD) of α7 nACHR and α4 nACHR in the seminiferous tubules and intertubular connective tissue in the MSG group compared to the control group. In the MSG+vitamin C group, the expression and OD of α7 nACHR and α4 nACHR increased in the seminiferous tubules and intertubular connective tissue but this improvement was not significant compared to the MSG group. MSG decreased the expression level of nACHR protein subunits, α7 and α4, in the seminiferous tubules and interstitial testicular tissue. Vitamin C in the MSG+vitamin C group could not significantly improve the expression of α7 and α4 nACHR subunits in testicular tissue. Probably, MSG toxicity can be compensated with higher doses of vitamin C.

GSK-J4 Inhibition of KDM6B Histone Demethylase Blocks Adhesion of Mantle Cell Lymphoma Cells to Stromal Cells by Modulating NF-κB Signaling.

Multiple signaling pathways facilitate the survival and drug resistance of malignant B-cells by regulating their migration and adhesion to microenvironmental niches. NF-κB pathways are commonly dysregulated in mantle cell lymphoma (MCL), but the exact underlying mechanisms are not well understood. Here, using a co-culture model system, we show that the adhesion of MCL cells to stromal cells is associated with elevated levels of KDM6B histone demethylase mRNA in adherent cells. The inhibition of KDM6B activity, using either a selective inhibitor (GSK-J4) or siRNA-mediated knockdown, reduces MCL adhesion to stromal cells. We showed that KDM6B is required both for the removal of repressive chromatin marks (H3K27me3) at the promoter region of NF-κB encoding genes and for inducing the expression of NF-κB genes in adherent MCL cells. GSK-J4 reduced protein levels of the RELA NF-κB subunit and impaired its nuclear localization. We further demonstrated that some adhesion-induced target genes require both induced NF-κB and KDM6B activity for their induction (e.g., IL-10 cytokine gene), while others require induction of NF-κB but not KDM6B (e.g., CCR7 chemokine gene). In conclusion, KDM6B induces the NF-κB pathway at different levels in MCL, thereby facilitating MCL cell adhesion, survival, and drug resistance. KDM6B represents a novel potential therapeutic target for MCL.

Upregulation of immunoproteasome PSMB8 is associated with Parkinson’s disease

BackgroundImmunoproteasome, a part of ubiquitin–proteasome system, is involved in immune response as well as protein degradation. However, the relationship between immunoproteasome and Parkinson's disease (PD) was not evaluated clearly. We hypothesized that the shift of immunoproteasome attributes to PD pathogenesis due to its role in inflammation and protein homeostasis. ObjectiveTo determine whether immunoproteasome in peripheral blood mononuclear cells (PBMC) and brain is expressed differently between patients with PD and healthy controls (HC). MethodsBlood samples were collected from 19 HC to 40 patients with PD of comparable ages. Peripheral blood mononuclear cells were isolated and followed by RT-qPCR to measure the mRNA levels of three catalytic subunits of immunoproteasome, namely, PSMB8, PSMB9, and PSMB10. Then, the protein levels of each subunit were measured by western blot. Finally, we confirmed the altered immunoproteasome subunit in the post-mortem human brain of PD. ResultsIn PBMCs, PSMB8 mRNA expression of PD group significantly increased compared to HC (p = 0.004), whereas PSMB9 and PSMB10 mRNA were not different between the PD and HC. The ratio of PSMB10 and PSMB8 mRNA (PSMB10/8 ratio) also reflected the significant difference between the PD and HC (p = 0.002). The PSMB10/8 ratio was well correlated with the UPDRS total and Part III score in the early stage of PD (Hoehn and Yahr ≤2.5) or drug-naïve PD subgroups. In terms of the protein level of immunoproteasome subunits in PBMCs, the increase of PSMB8 protein was observed in PD compared to HC (p = 0.0009), while PSMB9 and PSMB10 were not different between groups. Finally, we confirmed that immunoproteasome PSMB8 was expressed abundantly in the postmortem PD brain compared with normal control. ConclusionOur novel findings implicate that immunoproteasome PSMB8 is engaged in PD pathomechanism.

Expression changes of NaV channel subunits correlate with developmental maturation of electrophysiological characteristics of rat cerebellar Purkinje neurons

To investigate the variations in the expression of voltage-gated sodium (Nav) channel subunits during development of rat cerebellar Purkinje neurons and their correlation with maturation of electrophysiological characteristics of the neurons. We observed the changes in the expression levels of NaV1.1, 1.2, 1.3 and 1.6 during the development of Purkinje neurons using immunohistochemistry in neonatal (5-7 days after birth), juvenile (12-14 days), adolescent (21-24 days), and adult (42-60 days) SD rats. Using whole-cell patch-clamp technique, we recorded the spontaneous electrical activity of the neurons in ex vivo brain slices of rats of different ages to analyze the changes of electrophysiological characteristics of these neurons during development. The expression of NaV subunits in rat cerebellar Purkinje neurons showed significant variations during development. NaV1.1 subunit was highly expressed throughout the developmental stages and increased progressively with age (P < 0.05). NaV1.2 expression was not detected in the neurons in any of the developmental stages (P > 0.05). The expression level of NaV1.3 decreased with development and became undetectable after adolescence (P < 0.05). NaV1.6 expression was not detected during infancy, but increased with further development (P < 0.05). NaV1.1 and NaV1.3 were mainly expressed in the early stages of development. With the maturation of the rats, NaV1.3 expression disappeared and NaV1.6 expression increased in the neurons. NaV1.1 and NaV1.6 were mainly expressed after adolescence. The total NaV protein level increased gradually with development (P < 0.05) and tended to stabilize after adolescence. The spontaneous frequency and excitability of the Purkinje neurons increased gradually with development and reached the mature levels in adolescence. The developmental expression of NaV subunits was positively correlated with discharge frequency (r=0.9942, P < 0.05) and negatively correlated with the excitatory threshold of the neurons (r=0.9891, P < 0.05). The changes in the expression levels of NaV subunits are correlated with the maturation of high frequency electrophysiological properties of the neurons, suggesting thatmature NaV subunit expressions is the basis of maturation of electrophysiological characteristics of the neurons.

Sex differences in pituitary corticotroph excitability.

Stress-related illness represents a major burden on health and society. Sex differences in stress-related disorders are well documented, with women having twice the lifetime rate of depression compared to men and most anxiety disorders. Anterior pituitary corticotrophs are central components of the hypothalamic-pituitary-adrenal (HPA) axis, receiving input from hypothalamic neuropeptides corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP), while regulating glucocorticoid output from the adrenal cortex. The dynamic control of electrical excitability by CRH/AVP and glucocorticoids is critical for corticotroph function; however, whether corticotrophs contribute to sexually differential responses of the HPA axis, which might underlie differences in stress-related disorders, is very poorly understood. Using perforated patch clamp electrophysiology in corticotrophs from mice expressing green fluorescent protein under the control of the Pomc promoter, we characterized basal and secretagogue-evoked excitability. Both male and female corticotrophs show predominantly single-spike action potentials under basal conditions; however, males predominantly display spikes with small-amplitude (<20mV) afterhyperpolarizations (B-type), whereas females displayed a mixture of B-type spikes and spikes with a large-amplitude (>25mV) afterhyperpolarization (A-type). In response to CRH, or CRH/AVP, male cells almost exclusively transition to a predominantly pseudo-plateau bursting, whereas only female B-type cells display bursting in response to CRH±AVP. Treatment of male or female corticotrophs with 1nM estradiol (E2) for 24-72h has no effect on the proportion of cells with A- or B-type spikes in either sex. However, E2 results in the cessation of CRH-induced bursting in both male and female corticotrophs, which can be partially reversed by adding a BK current using a dynamic clamp. RNA-seq analysis of purified corticotrophs reveals extensive differential gene expression at the transcriptional level, including more than 71 mRNAs encoding ion channel subunits. Interestingly, there is a two-fold lower level (p < 0.01) of BK channel pore-forming subunit (Kcnma1) expression in females compared to males, which may partially explain the decrease in CRH-induced bursting. This study identified sex differences at the level of the anterior pituitary corticotroph ion channel landscape and control of both spontaneous and CRH-evoked excitability. Determining the mechanisms of sex differences of corticotroph and HPA activity at the cellular level could be an important step for better understanding, diagnosing, and treating stress-related disorders.

Potential of Quercetin to Protect Cadmium Induced Cognitive Deficits in Rats by Modulating NMDA-R Mediated Downstream Signaling and PI3K/AKT-Nrf2/ARE Signaling Pathways in Hippocampus.

Exposure to cadmium, a heavy metal distributed in the environment is a cause of concern due to associated health effects in population around the world. Continuing with the leads demonstrating alterations in brain cholinergic signalling in cadmium induced cognitive deficits by us; the study is focussed to understand involvement of N-Methyl-D-aspartate receptor (NMDA-R) and its postsynaptic signalling and Nrf2-ARE pathways in hippocampus. Also, the protective potential of quercetin, a polyphenolic bioflavonoid, was assessed in cadmium induced alterations. Cadmium treatment (5mg/kg, body weight, p.o., 28days) decreased mRNA expression and protein levels of NMDA receptor subunits (NR1, NR2A) in rat hippocampus, compared to controls. Cadmium treated rats also exhibited decrease in levels of NMDA-R associated downstream signalling proteins (CaMKIIα, PSD-95, TrkB, BDNF, PI3K, AKT, Erk1/2, GSK3β, and CREB) and increase in levels of SynGap in hippocampus. Further, decrease in protein levels of Nrf2 and HO1 associated with increase in levels of Keap1 exhibits alterations in Nrf2/ARE signalling in hippocampus of cadmium treated rats. Degeneration of pyramidal neurons in hippocampus was also evident on cadmium treatment. Simultaneous treatment with quercetin (25mg/kg body weight p.o., 28days) was found to attenuate cadmium induced changes in hippocampus. The results provide novel evidence that cadmium exposure may disrupt integrity of NMDA receptors and its downstream signaling targets by affecting the Nrf2/ARE signaling pathway in hippocampus and these couldcontribute in cognitive deficits. It is further interesting that quercetin has the potential to protect cadmium induced changes by modulating Nrf2/ARE signaling which was effective to control NMDA-R and PI3K/AKT cell signaling pathways.

Effect of chronic maternal L-Glu intake during gestation and/or lactation on oxidative stress markers, AMPA Glu1 receptor and adenosine A1 signalling pathway from foetal and neonatal cerebellum.

L-Glutamate (L-Glu) is an amino acid present in the diet that plays a fundamental role in the central nervous system, as the main excitatory neurotransmitter participating in learning and memory processes. In addition, the nucleoside adenosine has a crucial role in L-Glu metabolism, by regulating the liberation of this neurotransmitter through four different receptors: A1, A2A, A2B and A3, which activate (A2A and A2B) or inhibit (A1 and A3) adenylate cyclase pathway. L-Glu at high concentrations can act as a neurotoxin and induce oxidative stress. The study of the oxidative stress correlated with an excess of L-Glu consumption during maternity is key to understand its effects on foetuses and neonates. Previous studies have shown that there is a change in the receptor levels in the brain of pregnant rats and their foetuses when mothers are administered L-Glu during gestation; however, its effect on the cerebellum is unknown. Cerebellum is known to be responsible for motor, cognitive and emotional functions, so its possible involvement after L-Glu consumption is an important issue to study. Therefore, the aim of the present work was to study the effect of L-Glu exposure during gestation and lactation on oxidative stress biomarkers and neurotransmitter receptors from the cerebellum of foetuses and neonates. After maternal L-Glu intake during gestation, oxidative stress was increased, as the ionotropic L-Glu receptors, and GluR1 AMPA subunit levels were altered in foetuses. A1 adenosine receptor suffered changes after L-Glu treatment during gestation, lactation or both, in lactating neonate cerebellum, while adenylate cyclase activity remain unaltered. Further studies will be necessary to elucidate the importance of L-Glu intake and its possible excitotoxicity in the cerebellum of Wistar rats during the pregnancy period and their involvement in long-term neurodegeneration.

Cognitive dysfunction and impaired neuroplasticity following repeated exposure to the synthetic cannabinoid JWH-018 in male mice.

Psychotic disorders have been reported in long-term users of synthetic cannabinoids. This study aims at investigating the long-lasting effects of repeated JWH-018 exposure. Male CD-1 mice were injected with vehicle, JWH-018 (6mg·kg-1 ), the CB1 -antagonist NESS-0327 (1mg·kg-1 ) or co-administration of NESS-0327 and JWH-018, every day for 7days. After 15 or 16 days washout, we investigated the effects of JWH-018 on motor function, memory, social dominance and prepulse inhibition (PPI). We also evaluated glutamate levels in dialysates from dorsal striatum, striatal dopamine content and striatal/hippocampal neuroplasticity focusing on the NMDA receptor complex and the neurotrophin BDNF. These measurements were accompanied by in vitro electrophysiological evaluations in hippocampal preparations. Finally, we investigated the density of CB1 receptors and levels of the endocannabinoid anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and their main synthetic and degrading enzymes in the striatum and hippocampus. The repeated treatment with JWH-018 induced psychomotor agitation while reducing social dominance, recognition memory and PPI in mice. JWH-018 disrupted hippocampal LTP and decreased BDNF expression, reduced the synaptic levels of NMDA receptor subunits and decreased the expression of PSD95. Repeated exposure to JWH-018, reduced hippocampal CB1 receptor density and induced a long-term alteration in AEA and 2-AG levels and their degrading enzymes, FAAH and MAGL, in the striatum. Our findings suggest that repeated administration of a high dose of JWH-018 leads to the manifestation of psychotic-like symptoms accompanied by alterations in neuroplasticity and change in the endocannabinoid system.

Clinical analysis and functional characterization of KCNQ2-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathy (DEE) is a condition characterized by severe seizures and a range of developmental impairments. Pathogenic variants in KCNQ2, encoding for potassium channel subunit, cause KCNQ2-related DEE. This study aimed to examine the relationships between genotype and phenotype in KCNQ2-related DEE. In total, 12 patients were enrolled in this study for genetic testing, clinical analysis, and developmental evaluation. Pathogenic variants of KCNQ2 were characterized through a whole-cell electrophysiological recording expressed in Chinese hamster ovary (CHO) cells. The expression levels of the KCNQ2 subunit and its localization at the plasma membrane were determined using Western blot analysis. Seizures were detected in all patients. All DEE patients showed evidence of developmental delay. In total, 11 de novo KCNQ2 variants were identified, including 10 missense variants from DEE patients and one truncating variant from a patient with self-limited neonatal epilepsy (SeLNE). All variants were found to be loss of function through analysis of M-currents using patch-clamp recordings. The functional impact of variants on M-current in heteromericKCNQ2/3 channels may be associated with the severity of developmental disorders in DEE. The variants with dominant-negative effects in heteromeric channels may be responsible for the profound developmental phenotype. The mechanism underlying KCNQ2-related DEE involves a reduction of the M-current through dominant-negative effects, and the severity of developmental disorders in DEE may be predicted by the impact of variants on the M-current of heteromericKCNQ2/3 channels.

The role of protein kinase R in placental inflammation, mtUPR and apoptosis

IntroductionPlacental inflammation is implicated in the pathophysiology of many pregnancy complications, including fetal growth restriction, preeclampsia, gestational diabetes, and choriocarcinoma. Mitochondrial dysfunction, one of the outcomes of placental inflammation, is characterized by loss of membrane potential, accumulation of oxygen radicals, mitochondrial protein folding defects, and disturbances in mitochondrial dynamics. Protein kinase R (PKR) is stimulated by double-stranded RNA and bacterial endotoxins in the presence of pathogens and is a critical immune response enzyme. PKR is also correlated with the cell death response during endoplasmic reticulum stress. In this study, we aim to investigate the effects of PKR activity stimulated by lipopolysaccharide (LPS), and double-stranded RNA analog (Poly I:C) on mitochondrial unfolded protein response (mtUPR), mitochondrial membrane potential, apoptosis, and oxidative stress in placental trophoblasts. MethodsWe applied LPS and Poly I:C to BeWo cells to induce PKR activation. In addition, cells were treated with 2-aminopurine (2-AP) to inhibit the kinase activity of PKR. Protein levels of ATP-dependent Clp protease proteolytic subunit (CLPP) and heat shock protein 60 (HSP60) were determined after treatments. Apoptotic markers were detected by real-time PCR and flow cytometry. PKR-induced reactive oxygen radicals (ROS) accumulation and mitochondrial membrane potential change were assessed by flow cytometry. ResultsIt was determined that PKR activation-induced apoptosis in BeWo cells by reducing the levels of mtUPR proteins (CLPP and HSP60) and caused a decrease in mitochondrial membrane potential. PKR inhibition was sufficient for decreases in apoptotic markers and caused a reduction in the ratio of depolarized and ROS (+) cells. DiscussionOur results showed that LPS and Poly I:C administration stimulated PKR in BeWo cells in vitro. Furthermore, PKR activation is correlated with the levels of proteins involved in mitochondrial homeostasis and apoptosis. Our findings will contribute to understanding the role of PKR activation in placental inflammation and related diseases.

Age-dependent memory impairment induced by co-exposure to nicotine and a synthetic cannabinoid in mice

Co-use of marijuana and tobacco products is the second most common drug combination among adolescents. Nicotine (NIC) and cannabinoid use during adolescence induce similar detrimental changes, raising the hypothesis that simultaneous exposure could result in even more severe outcomes. Thus, we investigated whether the co-exposure to NIC and the synthetic cannabinoid WIN 55,212–2 (WIN) in adolescent mice causes behavioral outcomes different from those observed after exposure to a single drug. Male Swiss mice were exposed twice daily to NIC, WIN, or NIC + WIN during adolescence (PND28–47) or adulthood (PND70–89). Drug combination led to a greater reduction in weight gain in adolescent mice, while NIC-induced weight loss was observed in adults. During administration, NIC provoked hypothermia, and WIN produced hyperlocomotion in adolescent and adult mice. Animals exposed to NIC + WIN presented a profile of changes similar to those exposed to NIC. After drug exposure, changes in locomotion, thigmotaxis, social preference, prepulse inhibition, and working and recognition memory were evaluated. Adolescent but not adult mice exposed to NIC showed withdrawal-related hyperlocomotion unaffected by WIN co-administration. An age-specific impairment in object recognition memory was induced only by drug co-exposure during adolescence, which resolved spontaneously before reaching early adulthood. A transient decrease in hippocampal α7 nAChR subunit and CB1 receptor mRNA levels was induced by NIC exposure, which may be involved but is not enough to explain the memory impairment. Our work confirms the potential of NIC and cannabinoids association to aggravate some of the individual drug effects during critical neurodevelopmental periods.

N-acetylcysteine modulates rotenone-induced mitochondrial Complex I dysfunction in THP-1 cells

Mitochondrial Complex I dysfunction and oxidative stress have been part of the pathophysiology of several diseases ranging from mitochondrial disease to chronic diseases such as diabetes, mood disorders and Parkinson’s Disease. Nonetheless, to investigate the potential of mitochondria-targeted therapeutic strategies for these conditions, there is a need further our understanding on how cells respond and adapt in the presence of Complex I dysfunction. In this study, we used low doses of rotenone, a classical inhibitor of mitochondrial complex I, to mimic peripheral mitochondrial dysfunction in THP-1 cells, a human monocytic cell line, and explored the effects of N-acetylcysteine on preventing this rotenone-induced mitochondrial dysfunction. Our results show that in THP-1 cells, rotenone exposure led to increases in mitochondrial superoxide, levels of cell-free mitochondrial DNA, and protein levels of the NDUFS7 subunit. N-acetylcysteine (NAC) pre-treatment ameliorated the rotenone-induced increase of cell-free mitochondrial DNA and NDUFS7 protein levels, but not mitochondrial superoxide. Furthermore, rotenone exposure did not affect protein levels of the NDUFV1 subunit but induced NDUFV1 glutathionylation. In summary, NAC may help to mitigate the effects of rotenone on Complex I and preserve the normal function of mitochondria in THP-1 cells.

Effects of selective RNA processing and stabilization enzymes on carbon sequestration by photosynthesis of Synechococcus sp. PCC7002

Synechococcus is one of the most abundant prokaryotic photosynthetic organisms on Earth and plays a key role in oceanic carbon fixation and transformation. To improve the photosynthetic efficiency of synechococcus, a post-transcriptional regulatory mechanism - Selective RNA Processing and Stabilization (SRPS) was considered. We inactivated the SRPS-enzymes, executor of the SRPS mechanism, to explore their regulation rule of photosynthetic carbon fixation efficiency in Synechococcus. The results showed that the inactivation of SRPS-enzymes mainly affected the growth rate or growth phase. It significantly alters the photosynthetic oxygen evolution rate, pigment content, chlorophyll fluorescence, carbon and nitrogen content, as well as the composition and biological activity of the dissolved organic matter derived from Synechococcus (SOM). Inactivating SRPS-enzymes results in an increase in the expression level of most subunits of the Cytochrome b6-f complex, while the expression levels of most subunits of PSI, PSII, RuBisCO, and NDH decrease. All SRPS-enzymes are involved in the expression regulation of basilic protein complexes in photosynthesis, such as PSI, PSII, Cytochrome b6-f complex, ATP synthase, and RuBisCO. Our results indicate that the inactivation of SRPS-enzymes have a significant influence on carbon sequestration by photosynthesis of Synechococcus sp. PCC7002.

Prognostic significance of determining the activity and level of immune subunits of proteasomes in endometrial cancer

Введение. Рак эндометрия (РЭ) является одной из ведущей онкогинекологической патологией в России. В развитии рака эндометрия важную роль играют протеасомы – мультисубъединичные протеолитические комплексы. Цель исследования. Оценка прогностической значимости активности протеасом и уровня иммунных субъединиц протеасом LMP2 и LMP7 в отношении общей и безрецидивной выживаемости при раке эндометрия. Материалы и методы. В исследование было включено 90 больных раком эндометрия I-III стадии. Химотрипсинподобную активность (ХТА) протеасом определяли в опухолевой и неизмененной тканях эндометрия по гидролизу флуорогенного олигопептида Suc-LLVY-AMС (Sigma). Содержание иммунных субъединиц протеасом LMP2 и LMP7 определяли методом Вестерн блоттинг. Результаты. Выявлено увеличение химотрипсинподобной активности протеасом в опухолевой ткани по сравнению с неизмененным эндометрием, а также в опухолях III стадии по сравнению с I и II стадией. В ткани РЭ наблюдалось увеличение содержания LMP2 и LMP7 субъединиц по отношению к неизмененной ткани. Однофакторный анализ показателей протеасом показал, что с 3-летней безрецидивной выживаемостью больных раком эндометрия связана химотрипсинподобная активность протеасом, а с общей выживаемостью - содержание субъединицы LMP2 протеасом. Заключение. ХТА протеасом и содержание субъединицы LMP2 в опухолевой ткани можно рассматривать в качестве дополнительных критериев прогноза риска рецидивирования и общей выживаемости, что в свою очередь позволит планировать адекватный объем лечения больных.