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Journal Article 2015; 36(1): 7-14 PubMed PMID: 25789584 Citation  Keywords:  A Kinase Anchor Proteins:physiology, Animals, Humans, Signal Transduction:physiology,.   

OBJECTIVE: Kinase Anchoring Proteins (AKAPs) have evolved to regulate the spatial and temporal organization of cellular signal transduction. As a typical member, AKAP5 which consisting of three orthologues: bovine AKAP75, rodent AKAP150 and human AKAP79, is the best known model in the anchoring and targeting properties. It is shown that AKAP5 can bind β2-adrenergic receptor, which is a member of GPCR superfamily, and orchestrate the interactions of various protein kinases, protein phosphatases and cytoskeletal element. AKAP5 is originally identified as a component of the postsynaptic density in neurons and plays a vital role in modulating neuronal activities. Subsequently, the AKAP5 complexes are also detected in other tissues and participated in various processes....

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Journal Article 2014; 35(8): 684-690 PubMed PMID: 25702296 Citation  Keywords:  Animals, Apoptosis:drug effects, Dietary Supplements, Disease Models, Animal, Drug Therapy, Combination, Hippocampus:drug effects, Hormone Replacement Therapy, Hypothyroidism:drug therapy, Male, Oxidative Stress:drug effects, Random Allocation, Rats.Rats.   

OBJECTIVE: To examine the effect of levothyroxine (L-T4), vitamin E or both on oxidative stress status and hippocampal apoptosis in a propylthiouracil (PTU)-induced hypothyroid rat model.

METHODS: Sprague-Dawley rats were randomly divided into five groups: Control, PTU+PTU+L-T4+PTU+Vit E, PTU+Vit E+L-T4. In each group we assessed levels of serum triiodothyronine (T3), tetraiodothyronine (T4), thyroid stimulating hormone (TSH), hippocampus cellular apoptosis index (AI), hippocampus nicotinamide adenine denucleotide hydrogen (NADPH)oxidase and superoxide dismutase (SOD).

RESULTS: 1) Compared with the control group, NADPH oxidase levels were significantly increased, and SOD levels were significantly reduced in the PTU groups (p<0.05). 2) Compared to the PTU group, SOD levels were significantly increased in the PTU+Vit E and PTU+L-T4+Vit E group (p<0.05). NADPH oxidase levels were significantly decreased in the PTU+L-T4, PTU+Vit E and PTU+ L-T4+Vit E group (p<0.05). 3) Compared with the control group, hippocampus AI increased significantly in the PTU group (p<0.05). Compared with the PTU group, hippocampus AI was significantly reduced in the PTU+L-T4 group and PTU+L-T4+Vit E group (p<0.05). 4) Hippocampus AI was positively correlated with NADPH oxidase expression levels in hippocampus tissue (r=0.644, p<0.01).

CONCLUSION: Levothyroxine replacement therapy combined with vitamin E reduces hippocampus AI by improving oxidative stress. This study suggested that the mechanisms of hippocampus tissue injury in a hypothyroid rat model is related to hippocampus apoptosis from increased oxidative stress....

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Journal Article 2014; 35(Suppl 2): 169-174 PubMed PMID: 25638382 Citation  Keywords:  Depressive Disorder:complications, Female, Humans, Hypothalamo-Hypophyseal System:growth & development, Neurotransmitter Uptake Inhibitors:adverse effects, Pituitary-Adrenal System:growth & development, Postpartum Period:psychology, Pregnancy, Pregnancy C.   

: Depression during pregnancy and in the post partum period is a significant health issue in modern society. The estimated prevalence of depression in pregnancy ranges from 13-20%. The major dilemma for gynecologists is to treat or not to treat depression during gestation and lactation. Consequences of untreated depression can be so serious that the benefit of antidepressant therapy may overweigh the possible risk for injury of fetal/neonatal development. Currently, selective serotonin re-uptake inhibitors (SSRIs) and serotonin and noradrenaline re-uptake inhibitors (SNRIs) are commonly used for treatment of maternal depression. The review article brings up-to-date knowledge on effects of maternal adversity (depression) and/or antidepressants on the development of the hypothalamus-pituitary-adrenal axis of the offspring in relation to postnatal behavior and reactivity to stressful stimuli. Treated as well as untreated maternal depression presents a risk for the developing fetus and neonate. The authors stress the need to evaluate the relative safety of SNRIs/SNRIs by means of relevant experimental models to assess if these drugs can be assigned to treat pregnant and lactating depressive women.

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Journal Article 2014; 35(Suppl 2): 141-148 PubMed PMID: 25638378 Citation  Keywords:  Animals, Electron Transport Complex II:drug effects, Formazans:metabolism, Interferon-gamma:pharmacology, Mice, Mice, Inbred C57BL, Peritoneum:cytology, Pyrimidines:pharmacology, Spleen:cytology,.   

OBJECTIVES: Formation of formazan is a commonly used measure of cytotoxicity of compounds. It is a product of reduction of tetrazolium salts such as 4-[3- (4-iodophenyl)-2- (4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride. The extent of substrates reduction reflects the activity of enzymes succinate dehydrogenase (SDH; respiratory complex II) and lactate dehydrogenase (LDH), respectively. The aim of present study was a) to investigate formazan formation under the conditions of in vitro stimulation of cells with interferon-γ (IFN-γ) and lipopolysaccharide (LPS), and b) to analyse possible interference of pyrimidine analogues with formazan production.

METHODS: Peritoneal cells and splenocytes were obtained from C57BL/6 mice. They were cultured at 37 degrees C, 5% CO2 in humidified incubator. Levels of formazan were determined at the interval of 24 h of culture using the WST-1 and LDH assays. Nitric oxide (NO) was activated by IFN-γ plus LPS and assayed by Griess reagent 24 h afterwards. Pyrimidines were applied concomitantly with immunostimulatory agents.

RESULTS: IFN-γ enhanced concentration of SDH-produced formazan by macrophages (not by splenocytes) by approximately 50%. The activity of LDH remained unaffected. LPS was ineffective in both cases. While pyrimidines with NO-inhibitory properties suppressed the IFN-γ-enhanced levels of SDH-produced formazan, they did not change the LDH-dependent formazan production.

CONCLUSION: IFN-γ augments the SDH-produced formazan by macrophages. It does not change the LDH-dependent formazan formation. The enhancing effect may have a significant impact upon the appropriate interpretation of cytotoxic properties of drugs investigated under the conditions of immune stimulation of cells.

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Journal Article 2014; 35(Suppl 2): 114-122 PubMed PMID: 25638375 Citation  Keywords:  Animals, Aryl Hydrocarbon Hydroxylases:metabolism, Cell Membrane:metabolism, Cytochrome P450 Family 2, Cytochromes b5:metabolism,.   

OBJECTIVES: The mammalian mixed function oxidase (MFO) system participates in hydroxylation of many hydrophobic endogenous compounds as well as xenobiotics such as drugs and carcinogens. This biotransformation system, located in a membrane of endoplasmic reticulum, consists of cytochrome P-450 (P450), NADPH:P450 oxidoreductase and a facultative component, cytochrome b5. The knowledge of the interactions among the individual components of the MFO system is essential to understand the relationships between the structure and function of this system that finally dictate a qualitative and quantitative pattern of produced metabolites (e.g. detoxified xenobiotics and/or activated carcinogens). To elucidate the quantitative aspects of the interactions within the MFO system we acquired the photo-initiated cross-linking approach.

METHODS: The photo-initiated cross-linking employing cytochrome b5 as a protein nanoprobe [an amino acid analogue of methionine (pMet) was incorporated into cytochrome b5 sequence during recombinant expression] was used to quantify its interaction with P450 2B4 in a functional membrane complex. The cross-linking was initiated by UV-irradiation that formed from a pMet photolabile diazirine group highly reactive carbene biradical. This biradical is able to covalently bind amino acids in the close proximity and to form cross-link. The Met 96 of cytochrome b5 is situated in a linker region between its catalytic and membrane domains, while Met 126 and 131 are located in its membrane domain. The combination of several methods (electrophoresis in polyacrylamide gel, isoelectric focusing, Edman N-terminal degradation and amino acid analysis) was employed to characterize the molar ratio of P450 2B4 to cytochrome b5 in formed covalent cross-links to quantify their transient interactions.

RESULTS: The successfully produced cytochrome b5 nanoprobe (with confirmed pMet incorporation by mass spectrometry) stimulates the catalytical activity of P450 2B4 when reconstituted with NADPH:P450 oxidoreductase in vitro in dilauroylphosphatidylcholine (DLPC) vesicles. The cross-linking was carried out in similar reconstituted system without NADPH:P450 oxidoreductase, and at least three products were separated on 1D SDS-PAGE. The molar ratio of P450 to cytochrome b5 in each complex was estimated using the above-mentioned combination of methods as 1:1, 1:2 and 2:1.

CONCLUSIONS: The results demonstrate the utility of cytochrome b5 nanoprobe to study the interactions in MFO system. Using this nanoprobe, heterodimer with P450 2B4 and in addition also heterooligomers were identified, suggesting rather complex interactions of both proteins in this system that suppose the formation of such multimeric structures in the membrane of endoplasmic reticulum.

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Journal Article 2014; 35(7): 602-607 PubMed PMID: 25617883 Citation  Keywords:  Adult, Aged, Breast Neoplasms:metabolism, Carcinoma, Ductal, Breast:metabolism, Carcinoma, Lobular:metabolism, Female, Fibroadenoma:metabolism, Humans, Membrane Lipids:metabolism, Middle Aged, Neoplasm Staging, Oxidative Stress:physiology, Postoperative P.   

OBJECTIVES: To evaluate the level of oxidative damage to membrane lipids due to the breast cancer surgery in the early postoperative period.

PATIENTS AND METHODS: Blood samples were collected on the preoperative day and 24 hours postoperatively in 71 women operated for breast cancer, and preoperatively in 38 female patients with benign breast tumour. Lipid peroxidation (LPO) in the blood samples was estimated by measuring the concentrations of malondialdehyde+4-hydroxyalkenals (MDA+4-HDA) with spectrophotometry.

CLINICAL DATA INCLUDED: tumour site, tumour histological findings, cancer stage, grade, tumour volume, state of lymph nodes, type of surgery for breast, type of surgery for axilla.

RESULTS: Blood LPO level was similar in breast cancer patients and benign tumour patients (2.01±0.46 nmol/ml vs. 1.92±0.39 nmol/ml, respectively; p>0.05). In cancer patients, MDA+4-HDA increased on the first postoperative day, i.e. from 2.01±0.46 nmol/ml to 2.58±0.98 nmol/ml (p=0.0001). In women with benign breast tumour, LPO did not relate to the histological finding (p=0.8915). In the breast cancer group, preoperative LPO did not correlate with age, tumour volume and number of metastatic lymph nodes. Level of MDA+4-HDA was similar in stages I/II (2.03±0.46 nmol/ml) compared to stages III/IV (1.69±0.26 nmol/ml, p=0.1521). Consequently, levels of MDA+4-HDA did not relate to disease stage (p=0.1364).

CONCLUSIONS: Surgery for breast cancer causes peripheral increase in oxidative damage to macromolecules in the early postoperative period. Therefore, perioperative antioxidant supplementation should be considered....

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Journal Article 2014; 35(7): 577-585 PubMed PMID: 25617880 Citation  Keywords:  Adult, Antioxidants:metabolism, Autoimmune Diseases:immunology, Bacterial Translocation:immunology, Epitopes:immunology, Fatigue Syndrome, Chronic:immunology, Female, Humans, Immunoglobulin M:immunology, Male, Middle Aged, Nitroso Compounds:immunology, Ox.   

OBJECTIVES: There is evidence that inflammatory, oxidative and nitrosative stress (IO&NS) pathways participate in the pathophysiology of a subgroup of patients with Myalgic Encephalomyelitis/chronic fatigue syndrome (ME/CFS). Increased IgM-related autoimmune responses to oxidative specific epitopes (OSEs), including malondialdehyde (MDA), oleic acid and phosphatidyl inositol (Pi), and nitroso-(NO)-adducts, including NO-tryptophan (NOW), NO-arginine and NO-cysteinyl, are frequently observed in ME/CFS. Autoimmune responses in ME/CFS may be driven by increased bacterial translocation as measured by IgM and IgA responses to LPS of gram negative bacteria.

METHODS: The aim of this study is to examine whether IgM responses to OSEs and NO-adducts are related to a better outcome as measured by the Fibromyalgia and Fatigue Rating Scale (FF). 76 ME/CFS patients with initially abnormal autoimmune responses were treated with care-as-usual, including nutraceuticals with anti-IO&NS effects (NAIOS), such as L-carnitine, coenzyme Q10, taurine + lipoic acid, with or without curcumine + quercitine or N-acetyl-cysteine, zinc + glutamine.

RESULTS: We found that use of these NAIOS was associated with highly significant reductions in initially increased IgM-mediated autoimmune responses to OSEs and NO-adducts. A greater reduction in autoimmune responses to OSEs during intake of these NAIOS was associated with a lower FF score. Reductions in IgM responses to oleic acid, MDA and Pi, but not in any of the NO-adducts, were associated with reductions in severity of illness. These associations remained significant after adjusting for possible effects of increased bacterial translocation (leaky gut).

CONCLUSIONS: Our results show that autoimmune responses to OSEs are involved in the pathophysiology of ME/CFS and that these pathways are a new drug target in a subgroup of ME/CFS patients. Although hypernitrosylation and nitrosative stress play a role in ME/CFS, reductions in these pathways are not associated with lowered severity of illness. Randomized controlled trials with NAIOS should be carried out in the subgroup of ME/CFS patients with initially increased autoimmune responses to OSEs....

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Journal Article 2014; 35(6): 445-453 PubMed PMID: 25433843 Citation  Keywords:  Adult, Antibodies, Bacterial:blood, Bacterial Translocation:immunology, Cluster Analysis, Fatigue Syndrome, Chronic:complications, Female, Humans, Immunoglobulin A:blood, Immunoglobulin M:blood, Irritable Bowel Syndrome:complications, Lipopolysaccharides:.   

BACKGROUND: There is evidence that Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is accompanied by gastro-intestinal symptoms; and IgA and IgM responses directed against lipopolysaccharides (LPS) of commensal bacteria, indicating bacterial translocation.

METHODS: This study was carried out to examine gastro-intestinal symptoms in subjects with ME/CFS versus those with chronic fatigue (CF). The two groups were dissected by dichotomizing those fulfilling and not fulfilling Fukuda's critera. In these groups, we examined the association between gastro-intestinal symptoms and the IgA and IgM responses directed against commensal bacteria.

RESULTS: Using cluster analysis performed on gastro-intestinal symptoms we delineated that the cluster analysis-generated diagnosis of abdominal discomfort syndrome (ADS) was significantly higher in subjects with ME/CFS (59.6%) than in those with CF (17.7%). The diagnosis of ADS was strongly associated with the diagnosis of irritable bowel syndrome (IBS). There is evidence that ME/CFS consists of two subgroups, i.e. ME/CFS with and without ADS. Factor analysis showed four factors, i.e. 1) inflammation-hyperalgesia; 2) fatigue-malaise; 3) gastro-intestinal symptoms/ADS; and 4) neurocognitive symptoms. The IgA and IgM responses to LPS of commensal bacteria were significantly higher in ME/CFS patients with ADS than in those without ADS.

CONCLUSIONS: The findings show that ADS is a characteristic of a subset of patients with ME/CFS and that increased bacterial translocation (leaky gut) is associated with ADS symptoms. This study has defined a pathway phenotype, i.e bacterial translocation, that is related to ME/CFS and ADS/IBS and that may drive systemic inflammatory processes....

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