Maresin 1 attenuates neuroinflammation in a mouse model of perioperative neurocognitive disorders.

BACKGROUND: Resolution of inflammation is an active and dynamic process after surgery. Maresin 1 (MaR1) is one of a growing number of specialised pro-resolving lipids biosynthesised by macrophages that regulates acute inflammation. We investigated the effects of MaR1 on postoperative neuroinflammation, macrophage activity, and cognitive function in mice. METHODS: Adult male C57BL/6 (n=111) and Ccr2RFP/+Cx3cr1GFP/+ (n=54) mice were treated with MaR1 before undergoing anaesthesia and orthopaedic surgery. Systemic inflammatory changes, bone healing, neuroinflammation, and cognition were assessed at different time points. MaR1 protective effects were also evaluated using bone marrow derived macrophage cultures. RESULTS: MaR1 exerted potent systemic anti-inflammatory effects without impairing fracture healing. Prophylaxis with MaR1 prevented surgery-induced glial activation and opening of the blood-brain barrier. In Ccr2RFP/+Cx3cr1GFP/+ mice, fewer infiltrating macrophages were detected in the hippocampus after surgery with MaR1 prophylaxis, which resulted in improved memory function. MaR1 treatment also reduced expression of pro-inflammatory cell surface markers and cytokines by in vitro cultured macrophages. MaR1 was detectable in the cerebrospinal fluid of older adults before and after surgery. CONCLUSIONS: MaR1 exerts distinct anti-inflammatory and pro-resolving effects through regulation of macrophage infiltration, NF-κB signalling, and cytokine release after surgery. Future studies on the use of pro-resolving lipid mediators may inform novel approaches to treat neuroinflammation and postoperative neurocognitive disorders.

High warfarin sensitivity in carriers of CYP2C9*35 is determined by the impaired interaction with P450 oxidoreductase.

Cytochrome P450 2C9 (CYP2C9) metabolizes many clinically important drugs including warfarin and diclofenac. We have recently reported a new allelic variant, CYP2C9*35, found in a warfarin hypersensitive patient with Arg125Leu and Arg144Cys mutations. Here, we have investigated the molecular basis for the functional consequences of these polymorphic changes. CYP2C9.1 and CYP2C9-Arg144Cys expressed in human embryonic kidney 293 cells effectively metabolized both S-warfarin and diclofenac in NADPH-dependent reactions, whereas CYP2C9-Arg125Leu or CYP2C9.35 were catalytically silent. However, when NADPH was replaced by a direct electron donor to CYPs, cumene hydroperoxide, hereby bypassing the CYP oxidoreductase (POR), all variant enzymes were active, indicating unproductive interactions between CYP2C9.35 and POR. In silico analysis revealed a decrease of the electrostatic potential of CYP2C9-Arg125Leu-POR interacting surface and the loss of stabilizing salt bridges between these proteins. In conclusion, our data strongly suggest that the Arg125Leu mutation in CYP2C9.35 prevents CYP2C9-POR interactions resulting in the absence of NADPH-dependent CYP2C9-catalyzed activity in vivo, thus influencing the warfarin sensitivity in the carriers of this allele.

ERp29 regulates response to doxorubicin by a PERK-mediated mechanism.

ERp29 is an endoplasmic reticulum (ER) luminal protein with a putative secretion factor/escort chaperone function. Accumulated evidence has implicated ERp29 in the thyroglobulin secretion, polyoma virus transport and recently in carcinogenesis. ERp29 levels were elevated in the tumors of various origins and under the conditions of genotoxic stress, such as ionizing radiation. Here we report the induction of ERp29 during the treatment of cells with doxorubicin, a commonly used antineoplastic agent. Experiments in the p53 -/- cells and p53 knockout mouse revealed that doxorubicin effect on ERp29 is p53 dependent. The increase of ERp29 level appears to activate a negative feedback loop where the elevated amounts of ERp29 augment cell viability as shown by a clonogenic cell survival assay. To elucidate the mechanisms behind the doxorubicin effects we have studied the impact of ERp29 on the interaction with the ER stress-activated eukaryotic translation initiation factor 2-alpha kinase 3 (PERK) that was shown to facilitate tumor cells' resistance to drug toxicity. Co-immunoprecipitation demonstrated physical interaction of ERp29 with PERK and moreover, overexpression of ERp29 enhanced endogenous levels of PERK. Our results identify ERp29 as a novel regulator of PERK and provide evidence for the role of ER resident factors in the regulation of chemotherapeutic efficacy. These findings show that PERK may represent a nodal point between ER stress and chemotherapeutic response.

Unfolded protein response is involved in the pathology of human congenital hypothyroid goiter and rat non-goitrous congenital hypothyroidism.

The unfolded protein response (UPR) is an intracellular signaling pathway that regulates the protein folding and processing capacity of the endoplasmic reticulum (ER). The UPR is induced by the pharmacological agents that perturb ER functions but is also activated upon excessive accumulation of the mutant secretory proteins that are unable to attain correct three-dimensional structure and are thus retained in the ER. Such defects in intracellular protein transport underlie the development of a number of phenotypically diverse inherited pathologies, termed endoplasmic reticulum storage diseases (ERSD). We have studied UPR development in two similar ERSDs, human congenital goiter caused by the C1264R and C1996S mutations in the thyroglobulin (Tg) gene and non-goitrous congenital hypothyroidism in rdw dwarf rats determined by the G2320R Tg mutation. In both cases, these mutations rendered Tg incapable of leaving the ER. A major ER chaperone immunoglobulin-binding protein (BiP), and a novel putative escort chaperone endoplasmic reticulum protein 29 KDa (ERp29) were found to be associated with Tg, which might be interpreted as the contribution of the quality control machinery to the previously shown retention of Tg in the ER. We have extended our earlier observations of ER chaperone induction with the identification of the additional ER (ERp29, ERp72, calreticulin, protein disulfide isomerase (PDI)), cytoplasmic (heat shock protein (HSP)70, HSP90) and mitochondrial (mtHSP70) upregulated chaperones and folding enzymes. Activation of the transcriptional arm of UPR, as judged by the appearance of the spliced (active) form of X-box binding protein (XBP1) and processed activating transcription factor 6 (ATF6) transcription factors was suggested to contribute to the overexpression of the ER chaperones. The processing of ATF6 was observed in both human and rat tissues with Tg mutations. Whereas, in human tissues, weak splicing of XBP1 mRNA was detected only in the C1264R mutant, all rat thyroids including wild-type contained significant amounts of the spliced form of XBP1 as opposed to human liver and rat brain tissues, implying the existence of a previously unknown tissue-specific regulation of XBP1 processing.

Thioredoxin fold as homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa dimer.

BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.

Oligomerization properties of ERp29, an endoplasmic reticulum stress protein.

ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress-inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross-linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self-associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross-linking and immunoprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.

A stress-inducible rat liver endoplasmic reticulum protein, ERp29.

We have isolated, cDNA cloned and characterised a 29-kDa protein (ERp29), containing a C-terminal endoplasmic reticulum(ER)-retrieval signal, from the rat liver ER. ERp29 was induced to high levels in the rat hepatoma cells under metabolic stress conditions known to cause an aberrant accumulation of proteins in the ER [(e.g. culture in presence of the Ca2+ ionophore A23187, inhibitors of Ca2+-ATPase (thapsigargin), intracellular protein transport (brefeldin A), or protein N-glycosylation (tunicamycin)]. Experimental evidence of its localisation in the luminal compartment of the ER was obtained by topology studies including immunofluorescence microscopy, in vitro translation and proteinase protection assay. ERp29 constitutes about 0.1% of the rat hepatic microsomal proteins and is constitutively expressed in all rat tissues examined, as evident from northern blot analysis. In rat hepatoma cells ERp29 was found to be associated with the abundant molecular chaperone/stress protein BiP/GRP78 and this interaction was significantly enhanced after treatment with tunicamycin and A23187. Taken together, these results suggest that ERp29 is a member of the stress-response machinery of the ER.

Signal transduction-mediated activation of the aryl hydrocarbon receptor in rat hepatoma H4IIE cells.

We have investigated mechanisms of omeprazole (OME)-mediated induction of CYP1A1 and CYP3A, using the rat hepatoma H4IIE cell line, in comparison with mechanisms exerted by traditional aryl hydrocarbon receptor (AhR) ligands such as benso(a)pyrene (B(a)P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). OME did not bind specifically to AhR, and it could not activate the AhR complex in rat cytosol to a xenobiotic-responsive element (XRE)-binding form in vitro. Genistein, a tyrosine kinase inhibitor, and daidzein, an inhibitor of casein kinase II, efficiently inhibited OME-mediated but not B(a)P- or TCDD-mediated induction of CYP1A1, as monitored at the transcriptional, mRNA, and protein levels as well as by analysis of activation of XRE-luciferase reporter constructs transfected into H4IIE cells. The protease inhibitor Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) and lavendustin A also had similar OME-specific effects. In addition, insulin pretreatment caused an almost complete inhibition of OME-dependent CYP1A1 induction but only partially affected TCDD and B(a)P-mediated induction of CYP1A1. Staurosporine, an inhibitor of protein kinase C, impaired the induction by both B(a)P and OME. OME caused an approximately 2-fold increase in the level of CYP3A expression, but all inhibitors used were ineffective in preventing this induction. Gel shift analysis with radiolabeled XRE and specific peptide antibodies toward AhR and aryl hydrocarbon receptor nuclear translocator protein (Arnt) revealed an OME-mediated translocation of the AhR.Arnt complex into the nuclei. Genistein inhibited the specific nuclear XRE binding caused by OME, but it potentiated the formation of the TCDD-induced XRE.AhR complex. Although daidzein was able to effectively inhibit the OME-stimulated CYP1A1 gene transcription, it did not influence the OME-dependent AhR.XRE complex formation. The data are consistent with a mechanism for OME-mediated induction of CYP1A1 that involves activation of the AhR complex via intracellular signal transduction systems and that is distinct from induction mediated by AhR ligands.

Substrate-regulated, cAMP-dependent phosphorylation, denaturation, and degradation of glucocorticoid-inducible rat liver cytochrome P450 3A1.

The major rat glucocorticoid-inducible cytochrome P450 (CYP3A1) is known to be regulated at a transcriptional level by glucocorticoids and at a post-translational level by substrate-dependent stabilization. We have investigated mechanisms of substrate/ligand stabilization using primary hepatocytes, isolated liver microsomes from dexamethasone-treated rats, and purified enzymes. Treatment of hepatocytes with glucagon caused a 3-fold increase in CYP3A1 phosphorylation as well as an enhanced degradation rate of the enzyme. Specific CYP3A1 substrates or ligands, such as erythromycin, triacetyloleandomycin, and clotrimazole (CTZ) protected the enzyme from degradation in hepatocytes and inhibited in a concomitant manner (r = 0.99) glucagon-induced phosphorylation of the enzyme. In vitro experiments with purified CYP3A1 and isolated liver microsomes revealed one major site (Ser393) phosphorylated by the catalytic subunit of cAMP-dependent kinase, a reaction inhibited by ligands. Experiments in microsomes showed the presence of an endogenous cAMP-dependent kinase active on CYP3A1. Addition of exogenous cAMP-dependent kinase increased the rate of microsomal CYP3A1 phosphorylation, a reaction further stimulated by NADPH, but inhibited by CTZ. The microsomal phosphorylation caused a pronounced denaturation of cytochrome P450, as revealed spectrophotometrically, whereas CTZ protected from this reaction. Similar effects were noted when the CYP3A1-dependent 6 beta-hydroxylation of testosterone was monitored. It is suggested that the cellular CYP3A1 level is regulated to a significant extent posttranslationally by substrate-regulated cAMP-dependent phosphorylation on Ser393, followed by denaturation and degradation in the endoplasmic reticulum.

[The cerebral vasoconstrictor effects of malondialdehyde]. / Tserebral'nye vazokonstriktornye éffekty malonovogo dial'degida.

Malonic dialdehyde has a pronounced central vasoconstrictor effect, though when given in large doses, it causes a profound decrease in systemic blood pressure, however its vasoconstrictor effects are not directly related to fluctuations in systemic blood pressure. The universal antioxidant alpha-tocopherol acetate abolishes and prevents the central vasoconstrictor effects of malonic dialdehyde.

Hormone- and substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in the endoplasmic reticulum membranes.

We have examined differences in post-translational regulation between rat liver ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible CYP2B1 using hepatocyte cultures and subcellular fractions, prepared from starved and acetone-treated rats. The intracellular degradation of CYP2E1 was rapid (approximate t1/2 = 9 h) and increased by glucagon treatment of the cells in an isozyme-specific manner, whereas CYP2B1 degradation in the same cells, was slower (t1/2 = 21 h). The glucagon effect on CYP2E1 degradation was abolished by either cycloheximide treatment of cells, indicating the involvement of protein components with rapid turnover, or by lowering of the culture temperature to 23 degrees C. The rapid phase of CYP2E1 degradation was not influenced by inhibitors of the autophagosomal/lysosomal pathway. In vitro experiments with isolated liver microsomes revealed the presence of a Mg(2+)-ATP-activated proteolytic system active on CYP2E1, previously modified by phosphorylation on Ser-129 or denatured by reactive metabolites formed from carbon tetrachloride. Imidazole, a CYP2E1 substrate, specifically inhibited the rapid intracellular degradation of CYP2E1 and also prevented phosphorylation and subsequent proteolysis in isolated microsomes. In contrast, no proteolysis of CYP2B1 occurred under the conditions used. The microsomal Mg(2+)-ATP-dependent CYP2E1 proteolysis could not be solubilized with high salt and 0.05% sodium cholate, indicating the action of membrane-integrated protease(s). Subfractionation of microsomes revealed that the Mg(2+)-ATP-dependent proteolytic system active on CYP2E1 was present in both rough and smooth endoplasmic reticulum. It is suggested that hepatic cytochromes P450 are degraded both in a bulk process, according to the autophagosomal/lysosomal pathway and more rapidly, in a hormone- and substrate-regulated fashion, by a specific proteolytic system in the endoplasmic reticulum, active on physiologically or exogenously modified molecules.

[Papain phonophoresis in the treatment of suppurative wounds and inflammatory processes]. / Fonoforez papaina v lechenii gnoinykh ran i vospalitel'nykh protsessov.

The authors studied the effect of papain and dimethyl sulfoxide (DMSO) phonophoresis on the course of purulent wounds and inflammatory processes in patients. It is shown that the use of 1% papain solution together with DMSO by means of phonophoresis is a very effective and promising method for the treatment of purulent wounds and inflammatory infiltrates. The terms of restoration of the structure of the injured tissues are reduced by 1.8 times on the average.

A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone.

Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.

[Effect of x-irradiation on DNA-synthesis in uterine gland epithelium and the relationship of that effect to the phase of the mitotic cycle]. / Deistvie rentgenooblucheniia na sintez DNK v épitelii matochnykh zhelez i zavisimost' étogo deistviia ot fazy mitoticheskogo tsikla.

The autoradiographic method with the use of thymidone-3H was applied to the study of X-irradiation influence on the DNA synthesis in the epithelium of the endometrial glands of ovariectomized mice stimulated with synestrol, and of the dependence of this action on the mitotic cycle phase. It appeared that in local irradiation of the uterus with a dose of 400 r reduction of the labeled nuclei index (LNI) in the cells of the endometrial glands differed and depended on the mitotic cycle phase of the majority of the cells.