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Chronic low‐grade inflammation, particularly elevated tumor necrosis factor (TNF) levels, occurs due to advanced age and is associated with greater susceptibility to infection. One reason for this is age‐dependent macrophage dysfunction (ADMD). Herein, we use the adoptive transfer of alveolar macrophages (AM) from aged mice into the airway of young mice to show that inherent age‐related defects in AM were sufficient to increase the susceptibility to Streptococcus pneumoniae, a Gram‐positive bacterium and the leading cause of community‐acquired pneumonia. MAPK phosphorylation arrays using AM lysates from young and aged wild‐type (WT) and TNF knockout (KO) mice revealed multilevel TNF‐mediated suppression of kinase activity in aged mice. RNAseq analyses of AM validated the suppression of MAPK signaling as a consequence of TNF during aging. Two regulatory phosphatases that suppress MAPK signaling, Dusp1 and Ptprs, were confirmed to be upregulated with age and as a result of TNF exposure both ex vivo and in vitro. Dusp1 is known to be responsible for glucocorticoid‐mediated immune suppression, and dexamethasone treatment increased Dusp1 and Ptprs expression in cells and recapitulated the ADMD phenotype. In young mice, treatment with dexamethasone increased the levels of Dusp1 and Ptprs and their susceptibility to infection. TNF‐neutralizing antibody reduced Dusp1 and Ptprs levels in AM from aged mice and reduced pneumonia severity following bacterial challenge. We conclude that chronic exposure to TNF increases the expression of the glucocorticoid‐associated MAPK signaling suppressors, Dusp1 and Ptprs, which inhibits AM activation and increases susceptibility to bacterial pneumonia in older adults. [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of PF‐06650833, a highly potent and selective small‐molecule inhibitor of interleukin‐1–associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. Methods: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti–citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast‐like synoviocyte (FLS) cultures stimulated with Toll‐like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF‐06650833 was evaluated in vivo in the rat collagen‐induced arthritis (CIA) model and the mouse pristane‐induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple‐ascending‐dose clinical trial of PF‐06650833 were used to test in vivo human pharmacology. Results: In vitro, PF‐06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF‐06650833 reduced circulating autoantibody levels in the pristane‐induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF‐06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. Conclusion: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications. [ABSTRACT FROM AUTHOR]

Mammalian oocytes are arrested at the diplotene stage of prophase I during fetal or postnatal development. It was reported that cyclin‐dependent kinases (CDK1) was the sole CDK to drive the resumption of meiosis and CDK2 was dispensable for meiosis progression in mouse oocytes according to the conditional knockout studies. However, a recent study showed that CDK2 activity is essential for meiotic division and gametogenesis by means of gene‐directed mutagenesis, which avoids the compensatory activation of other CDKs. Taken the compensatory effect between CDKs after gene knockout, the physiological function of CDK2 activity in oocyte maturation remains unclear. To address this issue, we applied a specific small‐molecule inhibitor to restrain CDK2 activity transiently during oocyte meiotic maturation. Surprisingly, transient inhibition of CDK2 activity severely prevented the meiosis I completion although the meiotic resumption was not affected. Then we found that CDK2 activity was required for establishment of normal spindle and chromosome dynamics. Notably, CDK2 inhibition interrupted the anaphase‐promoting complex/cyclosome (APC/C)‐dependent degradation pathway by maintaining the activation of spindle assembly checkpoint (SAC). Interestingly, CDK2 inhibition prevented the egg activation as well. Overall, our data demonstrate that CDK2 kinase activity is required for proper dynamics of spindle and chromosomes, whose disturbance induces the continuous SAC activation and subsequent inactivation of APC/C activity in oocyte meiosis. [ABSTRACT FROM AUTHOR]

The extracts from Psoralea corylifolia Linn. (P. corylifolia) seeds have been shown to display antitumor activity. To date, the prospects of this plant and its active compounds in the treatment of non-small-cell lung cancer (NSCLC) have not been thoroughly studied. In this study, we identified a novel psorachromene compound that displays selective cytotoxic effects on all NSCLC cells tested, including NSCLC cells harboring epidermal growth factor receptor (EGFR) activation mutants (H1975L858R/T790M and H1975-MS35L858R/T790M/C797S ). Psorachromene induces G1 arrest in NSCLC cells harboring wild-type EGFR but induces apoptosis in NSCLC cells harboring activating EGFR mutations. Psorachromene inhibits activated EGFR signaling and kinase activity and suppresses tumor growth of implanted H1975-MS35L858R/T790M/C797S cells in nude mice. Molecular docking analysis revealed that psorachromene could form stronger bonds with mutant EGFR than wild-type EGFR, which might account for the greater cytotoxic effects observed in NSCLC cells harboring activating EGFR mutations (H1975 and H1975-MS35) than wild-type EGFR (A549). In conclusion, it is suggested that psorachromene is an attractive agent to be further explored for its use in the treatment of NSCLC patients harboring EGFR L858R/T790M/C797S. [ABSTRACT FROM AUTHOR]

Background and Purpose: Severe acute pancreatitis (SAP) is characterized by trypsinogen activation, infiltration of leucocytes and tissue necrosis but the intracellular signalling mechanisms regulating organ injury in the pancreas remain elusive. Rho-kinase is a potent regulator of specific cellular processes effecting several pro-inflammatory activities. Herein, we examined the role of Rho-kinase signalling in acute pancreatitis.Experimental Approach: Pancreatitis was induced by infusion of taurocholate into the pancreatic duct in C57BL/6 mice. Animals were treated with a Rho-kinase inhibitor Y-27632 (0.5-5 mg·kg⁻¹) before induction of pancreatitis.Key Results: Taurocholate infusion caused a clear-cut increase in blood amylase, pancreatic neutrophil infiltration, acinar cell necrosis and oedema formation in the pancreas. Levels of pancreatic myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), trypsinogen activation peptide (TAP) and lung MPO were significantly increased, indicating local and systemic disease. Inhibition of Rho-kinase activity dose-dependently protected against pancreatitis. For example, 5 mg·kg⁻¹ Y-27632 reduced acinar cell necrosis, leucocyte infiltration and pancreatic oedema by 90%, 89% and 58%, respectively, as well as tissue levels of MPO by 75% and MIP-2 by 84%. Moreover, Rho-kinase inhibition decreased lung MPO by 75% and blood amylase by 83%. Pancreatitis-induced TAP levels were reduced by 61% in Y-27632-treated mice. Inhibition of Rho-kinase abolished secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro.Conclusions and Implications: Our novel data suggest that Rho-kinase signalling plays an important role in acute pancreatitis by regulating trypsinogen activation and subsequent CXC chemokine formation, neutrophil infiltration and tissue injury. Thus, these results indicate that Rho-kinase may constitute a novel target in the management of SAP. [ABSTRACT FROM AUTHOR]

Mixed skeletal muscles of ICR/IAn mice contain 0.2% of the phosphorylase kinase activity found in C3H/He-mg mice at pH 8.2 in the presence of Ca2+. This lack of activity is caused by the absence of the normal phosphorylase kinase protein (Cohen, P. T. W. et al. (1976) Eur. J. Biocchem, 66, 347-356). The trace residual activity in ICR/IAn mice has quite different properties from the normal enzyme. It has a much higher activity ratio (pH 6.8/8.2), its activity is less dependent. on Ca2+ and it is not activated by cyclic- AMP-dependent protein kinase or by trypsin. Its elution behaviour on Sepharose 4B also differs from the normal enzyme. A consequence of the higher activity ratio (pH 6.8/8.2) and decreased effect of Ca2+, is that mixed skeletal muscles of ICR/IAn mice contain 7% of normal activity at pH 6.8 in the absence of Ca2+. The activity of phosphorylase kinase in C3H/He-mg control mice is sixfold lower in red oxidative (soleus) muscle and 15-fold lower in cardiac muscle than in mixed skeletal muscle. In contrast, the trace residual activity in ICR/lAn mice is present at the same level in soleus muscle, cardiac muscle and mixed skeletal muscle. Soleus muscles of ICR/lAn mice therefore contain 1.5% of normal activity at pH 8.2 in the presence of Ca2+ and 30% of normal activity at pH 6.8 in the absence of Ca2+. Cardiac muscle contains 6% of normal activity at pH 8.2 in the presence of Ca2+ and 60% of normal activity at pH 6.8 in the absence of Ca2+. When isolated soleus muscles were incubated with adrenalin, the level of phosphorylase a in ICR/IAn mice rose from 2.8% in the absence to 5.7% in the presence of the hormone. In contrast, the level of phosphorylase a in normal mice varied from 2.4% to 11.1% in the absence of adrenalin, and increased to 17.7-23.9% in the presence of the hormone. It is concluded that the trace phosphorylase kinase activity in ICR/lAn mice is capable of phosphorylating phosphorylase b in viva and that this enzyme is responsible for the very low level of phosphorylase a in resting soleus muscle of ICR/lAn mice, and perhaps even in normal mice. Since the residual enzyme cannot be activated by cyclic-AMP-dependent protein kinase, the elevation of phosphorylase a by adrenalin may be caused by an inhibition of phosphorylase phosphatase. The possibility that this occurs through the activation of protein phosphatase inhibitor 1 by cyclic-AMP-dependent protein kinase is discussed. Incubation of soleus muscles with adrenalin decreased the activity ratio (&minpl; glucose 6-phosphate) of glycogen synthase from 0.24 to 0.08 in ICR/IAn mice and 0.31 to 0.14 in C3H/He-mg or Swiss albino mice. These results demonstrate that the inactivation of glycogen synthase by adrenalin in resting soleus muscle does not result from the activation of phosphorylase kinase by cyclic-AMP-dependent protein kinase. It is most likely mediated by a direct phosphorylation catalysed by cyclic-AMP-dependent protein kinase, although the activation of protein phosphatase inhibitor I may also contribute to the effect. [ABSTRACT FROM AUTHOR]

Phosphorylase kinase from rabbit skeletal muscle has been shown to possess the structure (αβγδ)4, where the δ-subunit is identical to the calcium binding protein, termed calmodulin. The amount of calmodulin was found to be stoichiometric with the α, β and γ subunits in all preparations of phosphorylase kinase, and was not dissociated from the enzyme even in the presence of 8 M urea, provided that calcium ions were present. The activity of phosphorylase kinase was increased by the addition of calmodulin to the assay, and half-maximal activation was observed at a molar ratio, calmodulin/phosphorylase kinase, of 20:1. At saturating concentrations of calmodulin, all preparations of phosphorylase kinase had a specific activity of 13.5 ± 1.0 U/mg at pH 8.2. In the absence of calmodulin, the specific activity ranged from 2–7 U/mg, so that the stimulation by calmodulin varied from 2–7-fold with different preparations of phosphorylase kinase. The molecular basis for this variability is discussed. The stimulation of the activity by calmodulin was prevented by the addition of troponin-I or the antipsychotic drug trifluoperazine, whereas these compounds had little effect on the calcium-dependent activity in the absence of calmodulin. These results demonstrated that the activation by calmodulin was caused by the interaction of a second molecule of calmodulin with phosphorylase kinase. The existence of an additional calmodulin binding site was also indicated by the finding that phosphorylase kinase bound to calmodulin-Sepharose in the presence of calcium ions. Experiments with ICR/IAn mice which lack muscle phosphorylase kinase activity and C3H/He-mg mice with normal activity, demonstrated that when muscle extracts were fractionated with ammonium sulphate, 90–95% of the calmodulin which precipitated at 0-35% ammonium sulphate was bound to phosphorylase kinase. This information was used to show that at least 35% of the calmodulin in low ionic strength EDTA extracts of rabbit skeletal muscle was bound to phosphorylase kinase. Muscle extracts from ICR/IAn mice and C3H/He-mg mice had identical myosin light-chain kinase activities, showing that there was not a generalized defect in calmodulin-dependent enzymes in ICR/IAn mice. The skeletal muscle extracts of ICR/IAn mice contained 60% of the calmodulin found in C3H/He-mg mice, and addition of calmodulin did not restore phosphorylase kinase activity to muscle extracts prepared from ICR/IAn mice. This indicated that the lack of phosphorylase kinase activity was not caused by an absence of the calmodulin molecule. The role of calmodulin in the regulation of muscle phosphorylase kinase activity is discussed. [ABSTRACT FROM AUTHOR]

Background and Purpose: The δ-subunit-containing GABAA receptors, α4 β1 δ and α4 β3 δ, in dentate gyrus granule cells (DGGCs) are known to exhibit both spontaneous channel openings (i.e. constitutive activity) and agonist-induced current. The functional implications of spontaneous gating are unclear. In this study, we tested the hypothesis that constitutively active α4 β1/3 δ receptors limit agonist efficacy.Experimental Approach: Whole-cell electrophysiological recordings of adult male rat and mouse hippocampal DGGCs were used to characterize known agonists and antagonists at δ-subunit-containing GABAA receptors. To separate constitutive and agonist-induced currents, different recording conditions were employed.Key Results: Recordings at either 24°C or 34°C, including the PKC autoinhibitory peptide (19-36) intracellularly, removed spontaneous gating by GABAA receptors. In the absence of spontaneous gating, DGGCs responded to the α4 β1/3 δ orthosteric agonist Thio-THIP with a four-fold increased efficacy relative to recording conditions favouring constitutive activity. Surprisingly, the neutral antagonist gabazine was unable to antagonize the current by Thio-THIP. Furthermore, a current was elicited by gabazine alone only when the constitutive current was silenced (EC50 2.1 μM). The gabazine-induced current was inhibited by picrotoxin, potentiated by DS2, completely absent in δ-/- mice and reduced in β1-/- mice, but could not be replicated in human α4 β1/3 δ receptors expressed heterologously in HEK cells.Conclusion and Implications: Kinase activity infers spontaneous gating in α4 β1/3 δ receptors in DGGCs. This significantly limits the efficacy of GABAA agonists and has implications in pathologies involving aberrant excitability caused by phosphorylation (e.g. addiction and epilepsy). In such cases, the efficacy of δ-preferring GABAA ligands may be reduced. [ABSTRACT FROM AUTHOR]

Background: Autosomal recessive mutations in the glucocerebrosidase gene, Beta-glucocerebrosidase 1 (GBA1), cause the lysosomal storage disorder Gaucher's disease. Heterozygous carriers of most GBA1 mutations have dramatically increased Parkinson's disease (PD) risk, but the mechanisms and cells affected remain unknown. Glucocerebrosidase expression is relatively enriched in astrocytes, yet the impact of its mutation in these cells has not yet been addressed.Objectives: Emerging data supporting non-cell-autonomous mechanisms driving PD pathogenesis inspired the first characterization of GBA1-mutant astrocytes. In addition, we asked whether LRRK2, likewise linked to PD and enriched in astrocytes, intersected with GBA1 phenotypes.Methods: Using heterozygous and homozygous GBA1 D409V knockin mouse astrocytes, we conducted rigorous biochemical and image-based analyses of lysosomal function and morphology. We also examined basal and evoked cytokine response at the transcriptional and secretory levels.Results: The D409V knockin astrocytes manifested broad deficits in lysosomal morphology and function, as expected. This, however, is the first study to show dramatic defects in basal and TLR4-dependent cytokine production. Albeit to different extents, both the lysosomal dysfunction and inflammatory responses were normalized by inhibition of LRRK2 kinase activity, suggesting functional intracellular crosstalk between glucocerebrosidase and LRRK2 activities in astrocytes.Conclusions: These data demonstrate novel pathologic effects of a GBA1 mutation on inflammatory responses in astrocytes, indicating the likelihood of broader immunologic changes in GBA-PD patients. Our findings support the involvement of non-cell-autonomous mechanisms contributing to the pathogenesis of GBA1-linked PD and identify new opportunities to correct these changes with pharmacological intervention. © 2020 International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

Background and Purpose: Immunosuppressive drugs have shown great promise in treating autoimmune diseases in recent years. A series of novel oxazole derivatives were screened for their immunosuppressive activity. PO-322 [1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)] was identified as the most effective of these compounds. Here, we have investigated the mechanism(s) underlying the inhibition of T-cell proliferation in vitro by PO-322, as well as its effects on the delayed-type hypersensitivity (DTH) response and imiquimod-induced dermatitis in vivo.Experimental Approach: T-cell proliferation and apoptosis were analysed with flow cytometry. Cell viability was assessed with a CCK-8 assay. Protein kinase activity was assessed by SelectScreen Kinase Profiling Services. The phosphorylation of signal-regulated molecules was measured by Western blot. Cytokine levels were determined by elisa. The effect of PO-322 on DTH and imiquimod-induced dermatitis was evaluated in BALB/c mice.Key Results: PO-322 inhibited human T-cell proliferation with anti-CD3/anti-CD28 mAbs or alloantigen without significant cytotoxicity. Importantly, PO-322 was a selective inhibitor of the serum- and glucocorticoid-regulated kinase 1 (SGK1) and decreased NDRG1 phosphorylation but not p70S6K, STAT5, Akt, or ERK1/2 phosphorylation. Furthermore, PO-322 inhibited IFN-γ, IL-6, and IL-17 expression but not IL-10 expression. Finally, treatment with PO-322 was safe and effective for ameliorating the DTH response and imiquimod-induced dermatitis in mice.Conclusions and Implications: PO-322 exerted immunosuppressive activity in vitro and in vivo by selectively inhibiting SGK1 activity. PO-322 represents a potential lead compound for the design and development of new drugs for the treatment of autoimmune diseases. [ABSTRACT FROM AUTHOR]

Phosphatidylinositide 3-kinases (PI3Ks) are intracellular signal transducer enzymes that recruit protein kinase B (aka Akt) to the cell membrane, the subsequent activation of which regulates many cellular functions. PI3K/Akt activity is up-regulated within mesocorticolimbic structures in animal models of alcoholism, but less is known regarding PI3K/Akt activity in animal models of cocaine addiction. Given that prefrontal cortex (PFC) is grossly dysregulated in addiction, we studied how cocaine affects protein indices of PFC PI3K/Akt activity in rat and mouse models and examined the relevance of PI3K activity for cocaine-related learning. Immunoblotting of mouse medial PFC at 3 weeks withdrawal from a cocaine-sensitization regimen (seven injections of 30 mg/kg, intraperitoneal [IP]) revealed increased kinase activity, as did immunoblotting of tissue from the ventral PFC of rats with a history of long-access intravenous cocaine self-administration (0.25 mg/0.1 mL infusion; 10 days of 6 h/d cocaine access). Interestingly, increased Akt phosphorylation was observed in rat ventromedial PFC at both 3- and 30-day withdrawal only in animals re-exposed to cocaine-associated cues. A conditioned place-preference paradigm in mice and a cue-elicited drug-seeking test in rats were conducted to determine the functional relevance for elevated PI3K activity for addiction-related behavior. In both cases, an intra-PFC infusion of the PI3K inhibitor wortmannin (50μM) reduced drug-seeking behavior. Taken together, this cross-species, interdisciplinary, study provides convincing evidence that cocaine history produces an enduring increase in PI3K/Akt-dependent signaling within the more ventral aspect of the PFC that is relevant to behavioral reactivity to drug-associated cues/contexts. As such, PI3K inhibitors may well serve as an effective strategy for reducing drug cue reactivity and craving in cocaine addiction. [ABSTRACT FROM AUTHOR]

Background: Transient receptor potential vanilloid-1 (TRPV1), activated by heat, acidic pH, endogenous vanilloids and capsaicin, is essential for thermal hyperalgesia. Under inflammatory conditions, phosphorylation of TRPV1 by protein kinase C (PKC) can sensitize the channel and decrease the activation threshold. Src kinase also phosphorylates TRPV1, promoting channel trafficking to the plasma membrane. These post-translational modifications are important for several chronic pain conditions. This study presents a previously undescribed relationship between Src and PKC phosphorylation of TRPV1, influencing the thermal hypersensitivity associated with TRPV1 activation.Methods: We assessed TRPV1 channel activity using intracellular calcium imaging and patch-clamp electrophysiology in mouse dorsal root ganglion cultures. Additionally, we used behavioural experiments to evaluate plantar thermal sensitivity following intraplantar injections of activators of known modulators of TRPV1 with and without an Src antagonist.Results: Using calcium imaging and patch-clamp techniques, we demonstrated that pharmacological inhibition of Src kinase or mutation of the Src phosphorylation site on TRPV1 prevented PKC but not PKA-mediated sensitization of TRPV1 in vitro. We found that intraplantar injection of the PKC activator phorbol 12-myristate 13-acetate (PMA) or bradykinin produces thermal hypersensitivity that can be attenuated by pharmacological inhibition of Src. Additionally, complete Freund's Adjuvant (CFA)-induced inflammatory hypersensitivity could also be attenuated by local Src kinase inhibition.Conclusions: Our data demonstrate that Src phosphorylation is critical for PKC-mediated sensitization of TRPV1. Further, in a model of inflammatory pain, CFA, Src kinase inhibition could reduce thermal hypersensitivity. Targeting of Src kinase may have analgesic benefits in inflammatory pain conditions.Significance: Src kinase-mediated phosphorylation of TRPV1 is a critical regulator of the PKC-induced sensitization induced by multiple inflammatory mediators. This suggest a new regulatory mechanism governing TRPV1 function and a potential therapeutic target for inflammatory type pain, including cancer pain where Src antagonists are currently utilized. [ABSTRACT FROM AUTHOR]

Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by 35S-methionine/cysteine incorporation into cellular protein. Results: EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts . Conclusions: EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis. [ABSTRACT FROM AUTHOR]

Background and purpose:Mushrooms are popular both as food and as a source of natural compounds of biopharmaceutical interest. Some mushroom-derived compounds such as β-glucan have been shown to be immunostimulatory; this study explores the anti-inflammatory properties of hispidin analogues derived from the mushroom, Inonotus xeranticus. We sought to identify the molecular mechanism of action of these hispidin analogues by determining their effects on lipopolysaccharide (LPS)-mediated inflammatory responses in a macrophage cell line.Experimental approach:The production of inflammatory mediators was determined by Griess assay, reverse transcription-PCR and ELISA. The inhibitory effect of davalliactone on LPS-induced activation of signalling cascades was assessed by western blotting, immunoprecipitation and direct kinase assay.Key results:In activated RAW264.7 cells, davallialactone strongly downregulated LPS-mediated inflammatory responses, including NO production, prostaglandin E2 release, expression of proinflammatory cytokine genes and cell surface expression of co-stimulatory molecules. Davallialactone treatment did not alter cell viability or morphology. Davallialactone was found to exert its anti-inflammatory effects by inhibiting a signalling cascade that activates nuclear factor kappa B via PI3K, Akt and IKK, but not mitogen-activated protein kinases. Treatment with davallialactone affected the phosphorylation of these signalling proteins, but not their level of expression. These inhibitory effects were not due to the interruption of toll-like receptor 4 binding to CD14. In particular, davallialactone strongly inhibited the LPS-induced phosphorylation and kinase activity of Src, implying that Src may be a potential pharmacological target of davallialactone.Conclusions and implications:Our data suggest that davallialactone, a small molecule found in edible mushrooms, has anti-inflammatory activity. Davallialactone can be developed as a pharmaceutically valuable anti-Src kinase agent.British Journal of Pharmacology (2008) 154, 852–863; doi:10.1038/bjp.2008.136; published online 5 May 2008 [ABSTRACT FROM AUTHOR]

The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies. [ABSTRACT FROM AUTHOR]

Objective: Maternal high-fat diet (HFD) may alter the offspring intestinal immune system, thereby enhancing susceptibility toward inflammatory bowel disease. The objective of the current study was to investigate the impact of maternal HFD on offspring intestinal health using a mouse model of dextran sulfate sodium (DSS)-induced colitis.Methods: Dams were provided with either HFD (60%) or control diet. After weaning, female offspring from both groups were kept on 45% HFD. At 14 weeks of age, offspring were subjected to 2.5% DSS in drinking water for 5 days, followed by 5 days of recovery.Results: Offspring from maternal HFD had higher body weight gain before DSS induction and had higher liver and fat weights with increased adipocyte size at necropsy. When subjected to DSS treatment, HFD offspring had accelerated body weight loss and exaggerated disease activity index. HFD offspring had an elevated histopathological score and interleukin (IL)-1β, IL-6, and IL-17 expression with upregulated NF-κB signaling. Maternal HFD resulted in enhanced neutrophil infiltration associated with elevated expression of monocyte chemoattractant protein-1. Furthermore, maternal HFD suppressed AMP-activated protein kinase activity and decreased sirtuin 1 and p53 protein contents in offspring gut.Conclusions: Maternal HFD consumption predisposes offspring to a higher susceptibility to develop inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

We have previously reported [(1980) J. Biol. Chem. 255, 5999–6002] that retinoic acid inhibited growth and increased cyclic-AMP-dependent protein kinase activity in mouse melanoma cells. A variant melanoma line having depressed levels of cyclic-AMP-dependent protein kinase was not growth-inhibited by retinoic acid. In this report we describe the effect of retinoic acid on cyclic AMP binding proteins in B16 mouse melanoma cells. Using the technique of photoaffinity labeling, we found three major proteins of Mr 49000, 52000, and 55000 which were specifically labeled with 8-N3-[32P] AMP in both control and treated cells. Based upon their molecular weight, relative affinity for 8-N3-[32p]AMP and comigration with standards, we have designated the 49000-Mr and 55000-Mr species as RI and RII respectively. The position of the intermediate band (Mr 52000) was not affected by pre-incubation with ATP or alkaline phosphatase, and two-dimensional gel analysis indicated that it had the same pI as R1. Retinoic acid increased the 8-N3-[32p]AMP labeling of R1 within 24 h, reaching a maximal six fold increase by 48 h. These increases were limited to the 40000 × g supernatant fraction and occurred prior to any growth inhibition. By using increasing concentrations of 8-N3-cAMP we were able to construct a saturation curve for RI binding. Calculation of apparent Kd values from these curves showed nearly identical affinities for RI binding of 8-N3-cAMP from control and retinoic-acid-treated cells. Therefore we conclude that retinoic acid is increasing the amount of R1 rather than altering its properties. Corroboration of these results was obtained by DEAE-cellulose chromatography. Peak I (corresponding to type 1 protein kinase) from retinoid-treated cells was increased about six fold in binding activity. [ABSTRACT FROM AUTHOR]

Background We previously reported that acute functional tolerance ( AFT) to the hypnotic effects of alcohol was significantly correlated with drinking in the dark ( DID) in the LXS recombinant inbred panel, but only in mice that had been pretreated with alcohol. Here, we have conducted quantitative trait locus ( QTL) mapping for AFT. DNA sequencing of the progenitor ILS and ISS strains and microarray analyses were also conducted to identify candidate genes and functional correlates. Methods LXS mice were given either saline or alcohol (5 g/kg) on day 1 and then tested for loss of righting reflex AFT on day 2. QTLs were mapped using standard procedures. Two microarray analyses from brain were conducted: (i) naïve LXS mice and (ii) an alcohol treatment time course in the ILS and ISS. The full genomes of the ILS and ISS were sequenced to a depth of approximately 30×. Results A significant QTL for AFT in the alcohol pretreatment group was mapped to distal chromosome 4; numerous suggestive QTLs were also mapped. Preference drinking and DID have previously been mapped to the chromosome 4 locus. The credible interval of the significant chromosome 4 QTL spanned 23 Mb and included 716 annotated genes of which 150 had at least 1 nonsynonymous single nucleotide polymorphism or small indel that differed between the ILS and ISS; expression of 48 of the genes was cis-regulated. Enrichment analysis indicated broad functional categories underlying AFT, including proteolysis, transcription regulation, chromatin modification, protein kinase activity, and apoptosis. Conclusions The chromosome 4 QTL is a key region containing possibly pleiotropic genes for AFT and drinking behavior. Given that the region contains many viable candidates and a large number of the genes in the interval fall into 1 or more of the enriched functional categories, we postulate that many genes of varying effect size contribute to the observed QTL effect. [ABSTRACT FROM AUTHOR]

Random skin flaps have limited clinical application as a broad surgical reconstruction treatment because of distal necrosis. The prolyl hydroxylase domain‐containing protein inhibitor roxadustat (RXD) enhances angiogenesis and reduces oxidative stress and inflammation. This study explored the function of RXD in the survival of random skin flaps. Thirty‐six male Sprague–Dawley rats were randomly divided into low‐dose RXD group (L‐RXD group, 10 mg/kg/2 day), high‐dose RXD group (H‐RXD group, 25 mg/kg/2 day), and control group (1 mL of solvent, 1:9 DMSO:corn oil). The proportion of surviving flaps was determined on day 7 after surgery. Angiogenesis was assessed by lead oxide/gelatin angiography, and microcirculation blood perfusion was evaluated by laser Doppler flow imaging. Specimens in zone II were obtained, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as indicators of oxidative stress. Histopathological status was evaluated with haematoxylin and eosin staining. The levels of hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), and the inflammatory factors interleukin (IL)‐1β, IL‐6, and tumour necrosis factor‐α (TNF‐α) were detected by immunohistochemistry. RXD promoted flap survival and microcirculatory blood perfusion. Angiogenesis was detected distinctly in the experimental group. SOD activity increased and the MDA level decreased in the experimental group. Immunohistochemistry indicated that the expression levels of HIF‐1α and VEGF were increased while the levels of IL‐6, IL‐1β, and TNF‐α were decreased after RXD injection. RXD promoted random flap survival by reinforcing vascular hyperplasia and decreasing inflammation and ischaemia‐reperfusion injury. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 114, 203–214. Cyclin-dependent kinase 5/p35 kinase complex plays a critical role in dopaminergic neurotransmission. Dysregulation of dopamine (DA) signaling is associated with neurological and neuropsychiatric disorders. As cyclin-dependent kinase 5 (Cdk5) requires association with p35 for its proper activation, we hypothesized that dysregulation of Cdk5 activity might have an effect on striatal-mediated behavior. We used a mutant mouse, deficient in p35 protein (p35 KO), which displayed reduced Cdk5 activity. Throughout behavioral and biochemical characterization of naïve and psychostimulant-treated mice, we demonstrated that only juvenile p35 KO mice displayed spontaneous hyperactivity, responded with a paradoxical hypolocomotor effect to psychostimulant drugs and exhibited deficit on proper behavioral inhibition. Strong immunolabeling for tyrosine-hydroxylase and high striatal DA synthesis and contents with a low DA turnover, which were reverted by psychostimulants, were also found in mutant mice. Our results demonstrate that p35 deficiency is critically involved in the expression of a hyperactive behavioral phenotype with hyper-functioning of the dopaminergic system, emphasizing the importance of proper Cdk5 kinase activity for normal motor and emotional features. Thus, p35 KO mice may be another useful animal model for understanding cellular and molecular events underlying attention deficit hyperactivity disorder-like disorders. [ABSTRACT FROM AUTHOR]

Early onset increases in oxidative stress and tau pathology are present in the brain of senescence-accelerated mice prone (SAMP8). Astrocytes play an essential role, both in determining the brain's susceptibility to oxidative damage and in protecting neurons. In this study, we examine changes in tau phosphorylation, oxidative stress and glutamate uptake in primary cultures of cortical astrocytes from neonatal SAMP8 mice and senescence-accelerated-resistant mice (SAMR1). We demonstrated an enhancement of abnormally phosphorylated tau in Ser199 and Ser396 in SAMP8 astrocytes compared with that of SAMR1 control mice. Gsk3β and Cdk5 kinase activity, which regulate tau phosphorylation, was also increased in SAMP8 astrocytes. Inhibition of Gsk3β by lithium or Cdk5 by roscovitine reduced tau phosphorylation at Ser396. Moreover, we detected an increase in radical superoxide generation, which may be responsible for the corresponding increase in lipoperoxidation and protein oxidation. We also observed a reduced mitochondrial membrane potential in SAMP8 mouse astrocytes. Glutamate uptake in astrocytes is a critical neuroprotective mechanism. SAMP8 astrocytes showed a decreased glutamate uptake compared with those of SAMR1 controls. Interestingly, survival of SAMP8 or SAMR1 neurons cocultured with SAMP8 astrocytes was significantly reduced. Our results indicate that alterations in astrocyte cultures from SAMP8 mice are similar to those detected in whole brains of SAMP8 mice at 1–5 months. Moreover, our findings suggest that this in vitro preparation is suitable for studying the molecular and cellular processes underlying early aging in this murine model. In addition, our study supports the contention that astrocytes play a key role in neurodegeneration during the aging process. [ABSTRACT FROM AUTHOR]

Stimuli paired with reward acquire incentive properties that are important for many aspects of motivated behavior, such as feeding and drug-seeking. Here we used a novel chemical–genetic strategy to determine the role of the brain-derived neurotrophic factor (BDNF) receptor TrkB, known to be critical to many aspects of neural development and plasticity, during acquisition and expression of positive incentive value by a cue paired with food. We assessed that cue’s learned incentive value in a conditioned reinforcement task, in which its ability to reinforce instrumental responding later, in the absence of food itself, was examined. In TrkB F616A knock-in mice, TrkB kinase activity was suppressed by administering the TrkB inhibitor 1NMPP1 during the period of initial cue incentive learning only (i.e. Pavlovian training), during nose-poke conditioned reinforcement testing only, during both phases, or during neither phase. All mice acquired cue–food associations as indexed by approach responses. However, TrkB F616A mice that received 1NMPP1 during initial cue incentive learning failed to show conditioned reinforcement of nose-poking, regardless of their treatment in testing, whereas administration of 1NMMP1 only during the testing phase had no effect. The effects of 1NMPP1 administration were due to inhibition of TrkBF616A, because the performance of wild-type mice was unaffected by administration of the compound during either phase. These data indicate that BDNF or NT4 signaling through TrkB receptors is required for the acquisition of positive incentive value, but is not needed for the expression of previously acquired incentive value in the reinforcement of instrumental behavior. [ABSTRACT FROM AUTHOR]

To determine the activation status of two cytoplasmic signaling pathways, phosphatidylinositol 3-kinase (PI 3-kinase) and the mitogen-activated protein kinase (MAPK) family. We studied the pathogenic CD4+ T cells that drive disease in the parent-into-F1 mouse model of lupus-like chronic graft-versus-host disease (GVHD). We determined immunoprecipitated kinase activity for PI 3-kinase and MAPK members (Raf-1, extracellular signal–regulated kinase 1 [ERK-1], c-Jun N-terminal kinase 1 [JNK-1], and p38 MAPK) from either unfractionated splenocytes or purified donor CD4+ T cells. Uninjected normal mice served as negative controls, and acute GVHD mice served as positive controls. Compared with negative controls, unfractionated splenocyte kinase activity from chronic GVHD mice was significantly increased for PI 3-kinase and JNK-1, but not for Raf-1, p38 MAPK, or ERK-1. Increased PI 3-kinase and JNK-1 activity was also seen in acute GVHD splenocytes, as was increased Raf-1 and p38 MAPK activity. The pattern of increased PI 3-kinase and JNK-1 activity seen in unfractionated chronic GVHD splenocytes was also seen in isolated donor, but not host, CD4+ T cells from chronic GVHD mice, indicating that donor CD4+ T cell signaling activity accounted for at least a portion of the activity observed in unfractionated splenocytes. Increased ERK-1 activity was not seen in either donor or host CD4+ T cells. This pattern of cytoplasmic signaling pathway in donor CD4+ T cells was associated with increased T cell receptor membrane signaling activation (Lck and Fyn phosphorylation) and increased transcription activation (phosphorylation of inhibitor of nuclear factor κB), confirming the biologic significance of these observations. The pathogenic T cells driving disease in this murine model exhibit activation in the form of spontaneous cytoplasmic signaling pathway activity that can be detected without in vitro restimulation and involves a T cell–specific (PI 3-kinase) and a nonspecific stress/cytokine pathway (JNK-1). These results raise the possibility that a full characterization of the signaling pathways active in pathogenic lupus T cells might lead to new therapeutic targets. [ABSTRACT FROM AUTHOR]

1 The role of substance P and its high affinity neurokinin-1 receptor in colitis has not been fully elucidated. We assessed the participation of neurokinin-1 receptor in colitis using the 2,4,6,-trinitrobenzensulphonic add and dextran sulphate-induced animal models of colitis and genetically-engineered, neurokinin-1 receptor-deficient mice. 2 Clinical signs, macroscopic and histologic damage associated with 2,4,6,-trinitrobenzensulphonic acid (12 days) and dextran sulphate (5 days) colitis were more severe in neurokinin-1 deficient than in wild-type mice, while immuno reactivities for epidermal growth factor and its receptor were similar in the colon of both mice strains before and after colitis. 3 Substance P. dose-dependently induced intestinal fibroblast proliferation and enhanced epidermal growth factor-induced proliferation in intestinal fibroblasts isolated from wild-type, but not from neurokinin-1 receptor deficient mice. 4 Substance P-induced intestinal fibroblast proliferation required the presence of epidermal growth factor receptor with kinase activity. Furthermore, substance P induced epidermal growth factor tyrosine phosphorylation and activation in normal intestinal fibroblasts. 5 Our results indicate that in mice lacking the neurokinin-I receptor, substance P plays a protective role in prolonged experimental colitis. [ABSTRACT FROM AUTHOR]

Aim: Necroptosis participates in the pathogenesis of many inflammatory diseases, including periodontitis. Here, we aimed to investigate the role and mechanism of necroptosis inhibitors in attenuating periodontitis. Materials and Methods: The Gene Expression Omnibus (GEO) dataset GSE164241 was re‐analysed to identify the role of necroptosis in periodontitis. Gingival specimens from healthy subjects or periodontitis patients were collected to evaluate the expression level of necroptosis‐associated proteins. The therapeutic effect of necroptosis inhibitors on periodontitis was assessed in vivo and in vitro. Moreover, Transwell assays and Western blotting and siRNA transfection were used to identify the effects of necroptotic human gingival fibroblasts (hGFs) on THP‐1 macrophages. Results: Re‐analysis revealed that gingival fibroblasts (GFs) in periodontitis gingiva showed the highest area under the curve score of necroptosis. Elevated levels of necroptosis‐associated proteins were identified in GFs in periodontitis gingiva collected from patients and mice. In ligature‐induced periodontitis mice, local administration of receptor interacting protein kinase 3(RIPK3) inhibitor GSK′872 or sh‐mixed‐lineage kinase domain‐like pseudokinase (Mlkl) markedly abrogated necroptosis and rescued periodontitis. Analogously, necroptosis inhibitors alleviated the inflammatory response and release of damage‐associated molecular patterns in lipopolysaccharide‐ or LAZ (LPS + AZD'5582 + z‐VAD‐fmk, necroptosis inducer)‐induced GFs and then reduced THP‐1 cell migration and M1 polarization. Conclusions: Necroptosis in GFs aggravated gingival inflammation and alveolar bone loss. Necroptosis inhibitors attenuate this process by modulating THP‐1 macrophage migration and polarization. This study offers novel insights into the pathogenesis and potential therapeutic targets of periodontitis. [ABSTRACT FROM AUTHOR]

Down syndrome is the most common chromosomal abnormality in humans. Patients with Down syndrome have hematologic disorders, including mild to moderate thrombocytopenia. In case of Down syndrome, thrombocytopenia is not associated with bleeding, and it remains poorly characterized regarding molecular mechanisms. We investigated the effects of overexpression of Dyrk1A, an important factor contributing to some major Down syndrome phenotypes, on platelet number and bleeding in mice. Mice overexpressing Dyrk1A have a decrease in platelet number by 20%. However, bleeding time was found to be reduced by 50%. The thrombocytopenia and the decreased bleeding time observed were not associated to an abnormal platelet receptors expression, to a defect of platelet activation by ADP, thrombin or convulxin, to the presence of activated platelets in the circulation or to an abnormal half‐life of the platelets. To propose molecular mechanisms explaining this discrepancy, we performed a network analysis of Dyrk1A interactome and demonstrated that Dyrk1A, fibronectin and fibrinogen interact indirectly through two distinct clusters of proteins. Moreover, in mice overexpressing Dyrk1A, increased plasma fibronectin and fibrinogen levels were found, linked to an increase of the hepatic fibrinogen production. Our results indicate that overexpression of Dyrk1A in mice induces decreased bleeding consistent with increased plasma fibronectin and fibrinogen levels, revealing a new role of Dyrk1A depending on its indirect interaction with these two proteins. [ABSTRACT FROM AUTHOR]

Primary palatogenesis in mice is similar to that in humans, and spontaneous cleft lip appears to be multifactorially determined in both. Binding of a ligand to erbB4 has been shown to stimulate the receptor's protein kinase activity, which subsequently stimulates a signal-transduction cascade leading to cell growth and differentiation, and to morphogenesis during development. In this study, an immunohistochemical technique was used to investigate the temporal and spatial expression of erbB4 in the primary palate of cleft (A/WySn) and noncleft strains of mice (BALB/cBy). Positive staining of erbB4 was found in ectodermal and mesenchymal cells of facial prominences before the primary palate formation stage (day 10, hour 20) in both strains. During the primary palate formation stage (day 11, hour 20), positive staining of erbB4 was found in the ectodermal and mesenchymal cells of the facial prominences of the noncleft strain, but not in those of the cleft strain. These results suggest erbB4 expression may be associated with normal primary palatogenesis of mice and, conversely, cleft lip may be associated with a deficiency of erbB4 expression during primary palate formation in mice. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is a common neurodegenerative disease with limited treatment and no cure, hence, broadening PD drug spectrum is of great significance. At present, engineered microorganisms are attracting increasing attention. In this study, we constructed an engineered strain of Clostridium butyricum‐GLP‐1, a C. butyricum (a probiotic) that consistently expresses glucagon‐like peptide‐1 (GLP‐1, a peptide‐based hormone with neurological advantage) in anticipation of its use in PD treatment. We further investigated the neuroprotective mechanism of C. butyricum‐GLP‐1 on PD mice models induced by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine. The results indicated that C. butyricum‐GLP‐1 could improve motor dysfunction and ameliorate neuropathological changes by increasing TH expression and reducing the expression of α‐syn. Moreover, we confirmed that C. butyricum‐GLP‐1 improved microbiome imbalance of PD mice by decreasing the relative abundance of Bifidobacterium at the genus level, improved gut integrity, and upregulated the levels of GPR41/43. Surprisingly, we found it could exert its neuroprotective effects via promoting PINK1/Parkin mediated mitophagy and attenuating oxidative stress. Together, our work showed that C. butyricum‐GLP‐1 improves PD by promoting mitophagy, which provides an alternative therapeutic modality for PD. [ABSTRACT FROM AUTHOR]

Objective: Osteoarthritis (OA) is one of the most common degenerative joint diseases and is associated with autophagy suppression. However, the molecular mechanism of autophagy regulation in the context of OA is not fully understood. In this study, we sought to determine the role that HECTD1 plays in the pathogenesis of OA. Methods: We used RNA sequencing analysis to explore the differential expression of E3 ubiquitin ligase genes in healthy human cartilage and human cartilage affected by OA. Using surgery‐ and aging‐induced OA mouse models, we comprehensively analyzed the function of the screened gene Hectd1 in the development of OA; furthermore, we dissected the mechanism by which HECTD1 regulates autophagy and OA progression using a combination of molecular biologic, cell biologic, and biochemical approaches. Results: HECTD1 was significantly down‐regulated in human OA cartilage samples compared to healthy cartilage samples. Overexpression of HECTD1 in mouse joints alleviated OA pathogenesis, whereas conditional depletion of Hectd1 in cartilage samples aggravated surgery‐ and aging‐induced OA pathogenesis. Mechanistically, HECTD1 bound to Rubicon and ubiquitinated Rubicon at lysine residue 534, which targets Rubicon for proteasomal degradation. More importantly, HECTD1‐mediated Rubicon degradation regulated chondrocyte autophagy, leading to mitigation of stress‐induced chondrocyte death and the subsequent progression of OA. Conclusion: HECTD1 plays a crucial role in the pathogenesis of OA, in that HECTD1 regulates chondrocyte autophagy by ubiquitinating and targeting Rubicon for proteasomal degradation. [ABSTRACT FROM AUTHOR]

The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time‐lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI‐AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5 h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5 h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI‐AI transition by regulating the kinetochore‐microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes. [ABSTRACT FROM AUTHOR]

Objective: Itaconic acid, a Krebs cycle–derived immunometabolite, is synthesized by myeloid cells in response to danger signals to control inflammasome activation, type I interferon (IFN) responses, and oxidative stress. As these pathways are dysregulated in systemic lupus erythematosus (SLE), we investigated the role of an itaconic acid derivative in the treatment of established murine lupus. Methods: Female (NZW × NZB)F1 lupus‐prone mice were administered 4‐octyl itaconate (4‐OI) or vehicle starting after clinical onset of disease (30 weeks of age) for 4 weeks (n = 10 mice /group). At 34 weeks of age (peak disease activity), animals were euthanized, organs and serum were collected, and clinical, metabolic, and immunologic parameters were evaluated. Results: Proteinuria, kidney immune complex deposition, renal scores of severity and inflammation, and anti‐RNP autoantibodies were significantly reduced in the 4‐OI treatment group compared to the vehicle group. Splenomegaly decreased in the 4‐OI group compared to vehicle, with decreases in activation markers in innate and adaptive immune cells, increases in CD8+ T cell numbers, and inhibition of JAK1 activation. Gene expression analysis in splenocytes showed significant decreases in type I IFN and proinflammatory cytokine genes and increased Treg cell–associated markers in the 4‐OI group compared to the vehicle group. In human control and lupus myeloid cells, 4‐OI in vitro treatment decreased proinflammatory responses and B cell responses. Conclusions: These results support targeting immunometabolism as a potentially viable approach in autoimmune disease treatment, with 4‐OI displaying beneficial roles attenuating immune dysregulation and organ damage in lupus. [ABSTRACT FROM AUTHOR]

Background and Purpose: The pathogenesis of osteoarthritis implicates a low-grade inflammation associated to the innate immune system activation. Toll like receptor (TLR) stimulation triggers the release of inflammatory mediators, which aggravate osteoarthritis. We studied the preventive effect of 6-shogaol, a potential TLR4 inhibitor, on the treatment of experimental knee osteoarthritis.Experimental Approach: Osteoarthritis was induced in C57BL6 mice by surgical section of the medial meniscotibial ligament, which received 6-shogaol for eight weeks. Cartilage damage, inflammatory mediator presence and disease markers were assessed in joint tissues by immunohistochemistry. Computational modelling was used to predict binding modes of 6-shogaol into the TLR4/MD2 receptor and its permeability across cellular membranes. Employing LPS-stimulated chondrocytes and MAPK assay, we elucidated 6-shogaol action mechanisms.Key Results: 6-Shogaol treatment prevented articular cartilage lesions, synovitis and the presence of pro-inflammatory mediators, and disease markers in osteoarthritis animals. Molecular modelling studies predicted 6-shogaol interaction with the TLR4/MD-2 heterodimer in an antagonist conformation through its binding into the MD-2 pocket. In cell culture, we confirmed that 6-shogaol reduced LPS-induced TLR4 inflammatory signalling pathways. Besides, MAPK assay demonstrated that 6-shogaol directly inhibits the ERK1/2 phosphorylation activity.Conclusion and Implications: 6-Shogaol evoked a preventive action on cartilage and synovial inflammation in osteoarthritis mice. 6-shogaol effect may take place not only by hindering the interaction between TLR4 ligands and the TLR4/MD-2 complex in chondrocytes, but also through inhibition of ERK phosphorylation, implying a pleiotropic effect on different mediators activated during osteoarthritis, which proposes it as an attractive drug for osteoarthritis treatments. [ABSTRACT FROM AUTHOR]

Background and Purpose: Recent evidence links brain insulin resistance with neurodegenerative diseases, where hyperphosphorylated tau protein contributes to neuronal cell death. In the present study, we aimed to evaluate if d-pinitol inositol, which acts as an insulin sensitizer, affects the phosphorylation status of tau protein.Experimental Approach: We studied the pharmacological effect of d-pinitol on insulin signalling and tau phosphorylation in the hippocampus of Wistar and Zucker rats. To this end, we evaluated by western blotting the Akt pathway and its downstream proteins as being one of the main insulin-mediator pathways. Also, we explored the functional status of additional kinases phosphorylating tau, including PKA, ERK1/2, AMPK and CDK5. We utilized the 3xTg mouse model as a control for tauopathy, since it carries tau mutations that promote phosphorylation and aggregation.Key Results: Surprisingly, we discovered that oral d-pinitol treatment lowered tau phosphorylation significantly, but not through the expected kinase GSK-3 regulation. An extensive search for additional kinases phosphorylating tau revealed that this effect was mediated through a mechanism dependent on the reduction of the activity of the CDK5, affecting both its p35 and p25 subunits. This effect disappeared in leptin-deficient Zucker rats, uncovering that the association of leptin deficiency, obesity, dyslipidaemia and hyperinsulinaemia abrogates d-pinitol actions on tau phosphorylation. The 3xTg mice confirmed d-pinitol effectiveness in a genetic AD-tauopathy.Conclusion and Implications: The present findings suggest that d-pinitol, by regulating CDK5 activity through a decrease of CDK5R1, is a potential drug for developing treatments for neurological disorders such as tauopathies. [ABSTRACT FROM AUTHOR]

Salivary adenoid cystic carcinoma (SACC) is a malignant tumor, which is characterized by a higher incidence of distant metastasis. The aim of this study was to investigate the role and mechanism of protein kinase D1 (PKD1) in regulating the epithelial–mesenchymal transition (EMT) and promotes the metastasis in SACC. We analyzed the expression of PKD1 in 40 SACC patients and different metastatic potential cell lines. Then, we investigated whether the migration and growth of SACC were regulated by PKD1 using shRNA interference or inhibition of kinase active in vitro cell. Moreover, the mechanism by which PKD1 regulates the stability of Snail protein was determined. Finally, nude mice were used to testify the function of PKD1 via tail vein injection. PKD1 was correlated with metastasis and poor prognosis of SACC patients. PKD1 inhibition attenuated proliferation, migration, invasion, and EMT of SACC cells. Conversely, kinase active PKD1 could induce EMT and promoted cell migration in human HSG cell. Furthermore, downregulation of PKD1 regulated Snail via phosphorylation at Ser‐11 on Snail protein and promotion of proteasome‐mediated degradation, and reduced lung metastasis in vivo. Our results suggest that PKD1 induces the EMT and promotes the metastasis, which illustrate that PKD1 may be a potential prognostic biomarker and serve as a potential therapeutic target for SACC patients. [ABSTRACT FROM AUTHOR]

Objective: CR6‐interacting factor 1 (CRIF1) is a nuclear transcriptional regulator and a mitochondrial inner membrane protein; however, its functions in B lymphocytes have been poorly defined. This study was undertaken to investigate the effects of CRIF1 on B cell metabolic regulation, cell function, and autoimmune diseases. Methods: Using mice with B cell–specific deletion of CRIF1 (Crif1ΔCD19 mice), we assessed the relevance of CRIF1 function for lupus disease parameters, including anti–double‐stranded DNA (anti‐dsDNA), cytokines, and kidney pathology. RNA sequencing was performed on B cells from Crif1ΔCD19 mice. The phenotypic and metabolic changes in immune cells were evaluated in Crif1ΔCD19 mice. Roquinsan/+ mice crossed with Crif1ΔCD19 mice were monitored to assess the functionality of CRIF1‐deficient B cells in lupus development. Results: Crif1ΔCD19 mice showed an autoimmune lupus‐like phenotype, including high levels of autoantibodies to dsDNA and severe lupus nephritis with increased mesangial hypercellularity. While loss of CRIF1 in B cells showed impaired mitochondrial oxidative function, CRIF1‐deficient B cells promoted the production of interleukin‐17 (IL‐17) and IL‐6 and was more potent in helping T cells develop into follicular helper T cells. In a mouse model of autoimmune lupus, depletion of CRIF1 in B cells exacerbated lupus severity, and CRIF1 overexpression prevented lupus development in roquinsan/san mice. Conclusion: These results demonstrated that CRIF1 negatively correlates with disease severity and that overexpression of CRIF1 ameliorates disease development. Our findings suggest that CRIF1 is essential for preventing lupus development by maintaining B cell self tolerance. [ABSTRACT FROM AUTHOR]

Angiosarcomas are aggressive vascular sarcomas that arise from endothelial cells and have an extremely poor prognosis. Because of the rarity of angiosarcomas, knowledge of molecular drivers and optimized treatment strategies is lacking, highlighting the need for in vivo models to study the disease. Previously, we generated genetically engineered mouse models of angiosarcoma driven by aP2‐Cre‐mediated biallelic loss of Dicer1 or conditional activation of KrasG12D with Cdkn2a loss that histologically and genetically resemble human tumors. In the present study, we found that DICER1 functions as a potent tumor suppressor and its deletion, in combination with either KRASG12D expression or Cdkn2a loss, is associated with angiosarcoma development. Independent of the genetic driver, the mTOR pathway was activated in all murine angiosarcoma models. Direct activation of the mTOR pathway by conditional deletion of Tsc1 with aP2‐Cre resulted in tumors that resemble intermediate grade human kaposiform hemangioendotheliomas, indicating that mTOR activation was not sufficient to drive the malignant angiosarcoma phenotype. Genetic dissection of the spectrum of vascular tumors identified genes specifically regulated in the aggressive murine angiosarcomas that are also enriched in human angiosarcoma. The genetic dissection driving the transition across the malignant spectrum of endothelial sarcomas provides an opportunity to identify key determinants of the malignant phenotype, novel therapies for angiosarcoma, and novel in vivo models to further explore angiosarcoma pathogenesis. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Objective: IgG4‐related disease (IgG4‐RD) is a fibro‐inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7‐transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll‐like receptor 7 (TLR‐7)–positive CD163+ M2 macrophage infiltration in SGs from IgG4‐RD patients. We undertook this study to examine the fibrotic mechanism via the TLR‐7 pathway. Methods: Gene expression in SGs from human TLR7‐transgenic mice and IgG4‐RD patients was analyzed using DNA microarrays. We extracted the common up‐regulated TLR‐7–related genes in SGs from TLR7‐transgenic mice and IgG4‐RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR‐7 agonist loxoribine. Results: In TLR7‐transgenic mice and IgG4‐RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real‐time polymerase chain reaction validated the up‐regulation of only IRAK4 in IgG4‐RD patients compared with the other groups (P < 0.05). Interleukin‐1 receptor–associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4‐related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4‐positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P < 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF‐κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P < 0.05). Conclusion: CD163+ M2 macrophages promote fibrosis in IgG4‐RD by increasing the production of fibrotic cytokines via TLR‐7/IRAK4/NF‐κB signaling. [ABSTRACT FROM AUTHOR]

31Phosphorus magnetic resonance spectroscopy (31P‐MRS) has been shown to detect altered energetic status (e.g. the ratio of inorganic phosphate to phosphocreatine: Pi/PCr), intracellular acid–base status, and free intracellular magnesium ([Mg2+]) in dystrophic muscle compared with unaffected muscle; however, the causes of these differences are not well understood. The purposes of this study were to examine 31P‐MRS indices of energetic status and sarcolemma integrity in young mdx mice compared with wild‐type and to evaluate the effects of downhill running to induce muscle damage on 31P‐MRS indices in dystrophic muscle. In vivo 31P‐MRS spectra were acquired from the posterior hindlimb muscles in young (4–10 weeks of age) mdx (C57BL/10ScSn‐DMDmdx) and wild‐type (C57BL/10ScSnJ) mice using an 11.1‐T MR system. The flux of phosphate from PCr to ATP was estimated by 31P‐MRS saturation transfer experiments. Relative concentrations of high‐energy phosphates were measured, and intracellular pH and [Mg2+] were calculated. 1H2O‐T2 was measured using single‐voxel 1H‐MRS from the gastrocnemius and soleus using a 4.7‐T MR system. Downhill treadmill running was performed in a subset of mice. Young mdx mice were characterized by elevated 1H2O‐T2 (p < 0.01), Pi/PCr (p = 0.02), PCr to ATP flux (p = 0.04) and histological inflammatory markers (p < 0.05) and reduced (p < 0.01) [Mg2+] compared with wild‐type. Furthermore, 24 h after downhill running, an increase (p = 0.02) in Pi/PCr was observed in mdx and wild‐type mice compared with baseline, and a decrease (p < 0.001) in [Mg2+] and a lower (p = 0.048) intracellular [H+] in damaged muscle regions of mdx mice were observed, consistent with impaired sarcolemma integrity. Overall, our findings demonstrate that 31P‐MRS markers of energetic status and sarcolemma integrity are altered in young mdx compared with wild‐type mice, and these indices are exacerbated following downhill running. [ABSTRACT FROM AUTHOR]

cGMP interactors play a role in several pathologies and may be targets for cGMP analog‐based drugs, but the success of targeting depends on the biochemical stereospecificity between the cGMP‐analog and the interactor. The stereospecificity between general cGMP analogs—or such that are selectivity‐modified to obtain, for example, inhibitory actions on a specific target, like the cGMP‐dependent protein kinase—have previously been investigated. However, the importance of stereospecificity for cGMP‐analog binding to interactors is not known. We, therefore, applied affinity chromatography on mouse cortex proteins utilizing analogs with cyclic phosphate (8‐AET‐cGMP, 2‐AH‐cGMP, 2′‐AHC‐cGMP) and selectivity‐modified analogs with sulfur‐containing cyclic phosphorothioates (Rp/Sp‐8‐AET‐cGMPS, Rp/Sp‐2′‐AHC‐cGMPS) immobilized to agaroses. The results illustrate the cGMP analogs' stereospecific binding for PKG, PKA regulatory subunits and PKA catalytic subunits, PDEs, and EPAC2 and the involvement of these in various KEGG pathways. For the seven agaroses, PKG, PKA regulatory subunits, and PKA catalytic subunits were more prone to be enriched by 2‐AH‐, 8‐AET‐, Rp‐8‐AET‐, and Sp‐8‐AET‐cGMP, whereas PDEs and EPAC2 were more likely to be enriched by 2‐AH‐, Rp‐2′‐AHC‐, and Rp‐8‐AET‐cGMP. Our findings help elucidate the stereospecific‐binding sites essential for the interaction between individual cGMP analogs and cGMP‐binding proteins, as well as the cGMP analogs' target specificity, which are two crucial parameters in drug design. [ABSTRACT FROM AUTHOR]

Even though aberrant mechanistic target of rapamycin (mTOR) signaling is known to cause cardiomyopathy, its underlying mechanism remains poorly understood. Because augmentation of αB‐crystallin and hspB2 was presented in the cortical tubers and lymphangioleiomyomatosis of tuberous sclerosis complex patients, we deciphered the role of αB‐crystallin and its adjacent duplicate gene, hspB2, in hyperactive mTOR‐induced cardiomyopathy. Cardiac Tsc1 deletion (T1‐hKO) caused mouse mTOR activation and cardiomyopathy. Overexpression of αB‐crystallin and hspB2 was presented in the hearts of these mice. Knockout of αB‐crystallin/hspB2 reversed deficient Tsc1‐mediated fetal gene expression, mTOR activation, mitochondrial damage, cardiomyocyte vacuolar degeneration, cardiomyocyte size, and fibrosis of T1‐hKO mice. These cardiac‐Tsc1; αB‐crystallin; hspB2 triple knockout (tKO) mice had improved cardiac function, smaller heart weight to body weight ratio, and reduced lethality compared with T1‐hKO mice. Even though activated mTOR suppressed autophagy in T1‐hKO mice, ablation of αB‐crystallin and hspB2 failed to restore autophagy in tKO mice. mTOR inhibitors suppressed αB‐crystallin expression in T1‐hKO mice and rat cardiomyocyte line H9C2. Starvation of H9C2 cells activated autophagy and suppressed αB‐crystallin expression. Since inhibition of autophagy restored αB‐crystallin expression in starved H9C2 cells, autophagy is a negative regulator of αB‐crystallin expression. mTOR thus stimulates αB‐crystallin expression through suppression of autophagy. In conclusion, αB‐crystallin and hspB2 play a pivotal role in Tsc1 knockout‐related cardiomyopathy and are therapeutic targets of hyperactive mTOR‐associated cardiomyopathy. [ABSTRACT FROM AUTHOR]

Morphine is a potent opioid analgesic with high propensity for the development of antinociceptive tolerance. Morphine antinociception and tolerance are partially regulated by the midbrain ventrolateral periaqueductal gray (vlPAG). However, the majority of research evaluating mu‐opioid receptor signaling has focused on males. Here, we investigate kinase activation and localization patterns in the vlPAG following acute and chronic morphine treatment in both sexes. Male and female mice developed rapid antinociceptive tolerance to morphine (10 mg/kg i.p.) on the hot plate assay, but tolerance did not develop in males on the tail flick assay. Quantitative fluorescence immunohistochemistry was used to map and evaluate the activation of extracellular signal‐regulated kinase 1/2 (ERK 1/2), protein kinase‐C (PKC), and protein kinase‐A (PKA). We observed significantly greater phosphorylated ERK 1/2 in the vlPAG of chronic morphine‐treated animals which co‐localized with the endosomal marker, Eea1. We note that pPKC is significantly elevated in the vlPAG of both sexes following chronic morphine treatment. We also observed that although PKA activity is elevated following chronic morphine treatment in both sexes, there is a significant reduction in the nuclear translocation of its phosphorylated substrate. Taken together, this study demonstrates increased activation of ERK 1/2, PKC, and PKA in response to repeated morphine treatment. The study opens avenues to explore the impact of chronic morphine treatment on G‐protein signaling and kinase nuclear transport. [ABSTRACT FROM AUTHOR]

Bruton′s tyrosine kinase (Btk) is a crucial signaling molecule in BCR signaling and a key regulator of B‐ cell differentiation and function. Btk inhibition has shown impressive clinical efficacy in various B‐cell malignancies. However, it remains unknown whether inhibition additionally induces changes in BCR signaling due to feedback mechanisms, a phenomenon referred to as BCR rewiring. In this report, we studied the impact of Btk activity on major components of the BCR signaling pathway in mice. As expected, NF‐κB and Akt/S6 signaling was decreased in Btk‐deficient B cells. Unexpectedly, phosphorylation of several proximal signaling molecules, including CD79a, Syk, and PI3K, as well as the key Btk‐effector PLCγ2 and the more downstream kinase Erk, were significantly increased. This pattern of BCR rewiring was essentially opposite in B cells from transgenic mice overexpressing Btk. Importantly, prolonged Btk inhibitor treatment of WT mice or mice engrafted with leukemic B cells also resulted in increased phosho‐CD79a and phospho‐PLCγ2 in B cells. Our findings show that Btk enzymatic function determines phosphorylation of proximal and distal BCR signaling molecules in B cells. We conclude that Btk inhibitor treatment results in rewiring of BCR signaling, which may affect both malignant and healthy B cells. [ABSTRACT FROM AUTHOR]

Objective: The MAST family of microtubule-associated serine-threonine kinases (STKs) have distinct expression patterns in the developing and mature human and mouse brain. To date, only MAST1 has been conclusively associated with neurological disease, with de novo variants in individuals with a neurodevelopmental disorder, including a mega corpus callosum.Methods: Using exome sequencing, we identify MAST3 missense variants in individuals with epilepsy. We also assess the effect of these variants on the ability of MAST3 to phosphorylate the target gene product ARPP-16 in HEK293T cells.Results: We identify de novo missense variants in the STK domain in 11 individuals, including 2 recurrent variants p.G510S (n = 5) and p.G515S (n = 3). All 11 individuals had developmental and epileptic encephalopathy, with 8 having normal development prior to seizure onset at <2 years of age. All patients developed multiple seizure types, 9 of 11 patients had seizures triggered by fever and 9 of 11 patients had drug-resistant seizures. In vitro analysis of HEK293T cells transfected with MAST3 cDNA carrying a subset of these patient-specific missense variants demonstrated variable but generally lower expression, with concomitant increased phosphorylation of the MAST3 target, ARPP-16, compared to wild-type. These findings suggest the patient-specific variants may confer MAST3 gain-of-function. Moreover, single-nuclei RNA sequencing and immunohistochemistry shows that MAST3 expression is restricted to excitatory neurons in the cortex late in prenatal development and postnatally.Interpretation: In summary, we describe MAST3 as a novel epilepsy-associated gene with a potential gain-of-function pathogenic mechanism that may be primarily restricted to excitatory neurons in the cortex. ANN NEUROL 2021;90:274-284. [ABSTRACT FROM AUTHOR]

Hematopoiesis is a continuous phenomenon involving the formation of hematopoietic stem cells (HSCs) giving rise to diverse functional blood cells. This developmental process of hematopoiesis is evolutionarily conserved, yet comparably different in various model organisms. Vertebrate HSCs give rise to all types of mature cells of both the myeloid and the lymphoid lineages sequentially colonizing in different anatomical tissues. Signal transduction in HSCs facilitates their potency and specifies branching of lineages. Understanding the hematopoietic signaling pathways is crucial to gain insights into their deregulation in several blood‐related disorders. The focus of the review is on hematopoiesis corresponding to different model organisms and pivotal role of indispensable hematopoietic pathways. We summarize and discuss the fundamentals of blood formation in both invertebrate and vertebrates, examining the requirement of key signaling nexus in hematopoiesis. Knowledge obtained from such comparative studies associated with developmental dynamics of hematopoiesis is beneficial to explore the therapeutic options for hematopoietic diseases. HIGHLIGHTS: Model organisms exhibit multiple waves of hematopoiesis in different stages of development.Developmental dynamics of hematopoiesis is an intricate network of balance between blood cells and signaling pathways.Previous and on‐going studies on hematopoiesis in different model organisms contribute to the significance of signal transduction associated with blood cell development.Multiple signaling pathways foster the formation and development of distinct hematopoietic lineages and the aberrant signaling results in defective hematopoiesis. [ABSTRACT FROM AUTHOR]

Insulin resistance, a smaller than expected response to a given dose of insulin, is associated with many common diseases including, ageing, polycystic ovarian disease, syndrome X, cancer, infections, trauma and, most significantly, obesity and type 2 diabetes mellitus. The biochemical basis of insulin resistance in type 2 diabetes has been the subject of many studies. Earlier studies have indicated that quantitative regulation of the insulin sensitive glucose transporters (Glut-4) and insulin receptors themselves may contribute to this disorder, however, these two factors are probably inadequate to explain the extent of insulin resistance. This point also became apparent by the development of only mild hyperinsulinaemia in mice with a targeted mutation in the Glut-4 gene. Studies on postreceptor defects in type 2 diabetes has recently focused on the intrinsic catalytic activity of the insulin receptor and downstream signalling events. A reduction in tyrosine phosphorylation of both the insulin receptor (IR) and the insulin receptor substrate-1 (IRS-1) has been noted in both animal and human type 2 diabetes. Importantly, this appears to occur in all of the major insulin-sensitive tissues, namely the muscle, fat and liver. It is now clear that decreased signalling capacity of the insulin receptor is an important component of this disease. I will review some of the potential mechanisms underlying this deficiency. [ABSTRACT FROM AUTHOR]

γ‐Aminobutyric acid (GABA) acting through heteropentameric GABAA receptors plays a pivotal role in the sleep‐promoting circuitry. Whereas the role of the different GABAA receptor α‐subunits in sleep regulation and in mediating the effect of benzodiazepines for treatment of insomnia is well‐described, the β‐subunits are less studied. Here we report the first study characterizing sleep in mice lacking the GABAA receptor β1‐subunit (β1−/− mice). We show that β1−/− mice have a distinct and abnormal sleep phenotype characterized by increased delta power in non‐rapid eye movement (NREM) sleep and decreased theta activity in rapid eye movement (REM) sleep compared to β1+/+ mice, without any change in the overall sleep‐wake architecture. From GABAA receptor‐specific autoradiography, it is further demonstrated that functional β1‐subunit‐containing GABAA receptors display the highest binding levels in the hippocampus and frontal cortex. In conclusion, this study suggests that the GABAA receptor β1‐subunit does not play an important role in sleep initiation or maintenance but instead regulates the power spectrum and especially the expression of theta rhythm. This provides new knowledge on the complex role of GABAA receptor subunits in sleep regulation. In addition, β1−/− mice could provide a useful mouse model for future studies of the physiological role of delta and theta rhythms during sleep. [ABSTRACT FROM AUTHOR]

Four monoclonal antibodies (mAbs), derived from high-responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype γ1, κ and 15G4 is of isotype γ2b, κ. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific for the bovine γ isoform of protein kinase C and cross-react with protein kinase Cγ from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase Cγ cofactordependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor-independent catalytic activity of protein kinase Cγ. Competition analysis between these mAbs and phosphatidylserine, 12-O-tetradecanoylphorbol 13-acetate and Ca2+ showed that 36G9 and 5A2 block cofactor binding to protein kinase Cγ. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase Cγ. [ABSTRACT FROM AUTHOR]

Background and Purpose: Kaempferol is a natural flavonoid widely investigated in various fields due to its antioxidant, anti-cancer, and anti-inflammatory activities, but few studies have shown its inhibitory effect on T cell activation. This study examined the therapeutic potential of kaempferol in atopic dermatitis by modulating T cell activation.Experimental Approach: Effects of kaempferol on T cell activation and the underlying mechanisms were investigated in Jurkat cells and mouse CD4+ T cells. A model of atopic dermatitis in mice was used to determine its therapeutic potential on T cell-mediated conditions in vivo. Western blots, RT-PCR, pulldown assays and ELISA were used, along with histological analysis of skin.Key Results: Pretreatment with kaempferol reduced CD69 expression and production of inflammatory cytokines including IL-2 from activated Jurkat cells and murine CD4+ T cells without cytotoxicity. Pulldown assays revealed that kaempferol physically binds to MRP-1 in T cells, inhibiting the action of MRP-1. In activated T cells, kaempferol suppressed JNK phosphorylation and the TAK1-IKKα mediated NF-κB pathway. Oral administration of kaempferol to mice showed improved manifestation of atopic dermatitis, a T cell-mediated condition. Western blot results showed that, as in the in vitro studies, decreased phosphorylation of JNK was associated with down-regulated MRP-1 activity in vivo, in the kaempferol-treated mice in the atopic dermatitis model.Conclusion and Implications: Kaempferol regulates T cell activation by inhibiting MRP-1 activity in activated T cells, thus showing protective effects against T cell mediated disease in vivo. [ABSTRACT FROM AUTHOR]

Ptk2b has been found playing critical roles in oocyte maturation and subsequent fertilization in vitro. But what is the exact in vivo function in reproduction still elusive. Here, by constructing Ptk2b mutant mice, we found Ptk2b was not essential for mice fertility, unexpectedly, contrary to previously reported in vitro findings, we found Ptk2b ablation significantly improved female fecundity. Follicle counting indicated that the number of primordial follicles and growing follicles in matured mice was significantly increased in the absence of Ptk2b, whereas the primordial follicle formation showed no defects. We also found this regulation was in an autophosphorylation independent pathway, as autophosphorylation site mutant mice (PTK2BY402F) show no phenotype in female fertility. Further biochemistry studies revealed that Ptk2b ablation promotes folliculogenesis via Erk pathway mediate follicle survival. Together, we found a novel biological function of Ptk2b in folliculogenesis, which could be potentially used as a therapeutic target for corresponding infertility. [ABSTRACT FROM AUTHOR]

Liver steatosis is associated with increased ischaemia reperfusion (I/R) injury. Our previous studies have shown that irisin, an exercise‐induced hormone, mitigates I/R injury via binding to αVβ5 integrin. However, the effect of irisin on I/R injury in steatotic liver remains unknown. Kindlin‐2 directly interacts with β integrin. We therefore suggest that irisin protects against I/R injury in steatotic liver via a kindlin‐2 dependent mechanism. To study this, hepatic steatosis was induced in male adult mice by feeding them with a 60% high‐fat diet (HFD). At 12 weeks after HFD feeding, the mice were subjected to liver ischaemia by occluding partial (70%) hepatic arterial/portal venous blood for 60 minutes, which was followed by 24 hours reperfusion. Our results showed HFD exaggerated I/R‐induced liver injury. Irisin (250 μg/kg) administration at the beginning of reperfusion attenuated liver injury, improved mitochondrial function, and reduced oxidative and endoplasmic reticulum stress in HFD‐fed mice. However, kindlin‐2 inhibition by RNAi eliminated irisin's direct effects on cultured hepatocytes. In conclusion, irisin attenuates I/R injury in steatotic liver via a kindlin‐2 dependent mechanism. [ABSTRACT FROM AUTHOR]