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Werner EG Müller, Meik Neufurth, Shunfeng Wang, Heinz C Schröder, Xiaohong Wang ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, GermanyCorrespondence: Werner EG Müller; Xiaohong Wang, ERC Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, Mainz, D-55128, Germany, Tel +49 6131 392 5791, Email wmueller@uni-mainz.de; wang013@uni-mainz.deAbstract: There is a need for novel nanomaterials with properties not yet exploited in regenerative nanomedicine. Based on lessons learned from the oldest metazoan phylum, sponges, it has been recognized that two previously ignored or insufficiently recognized principles play an essential role in tissue regeneration, including biomineral formation/repair and wound healing. Firstly, the dependence on enzymes as a driving force and secondly, the availability of metabolic energy. The discovery of enzymatic synthesis and regenerative activity of amorphous biosilica that builds the mineral skeleton of siliceous sponges formed the basis for the development of successful strategies for the treatment of osteochondral impairments in humans. In addition, the elucidation of the functional significance of a second regeneratively active inorganic material, namely inorganic polyphosphate (polyP) and its amorphous nanoparticles, present from sponges to humans, has pushed forward the development of innovative materials for both soft (skin, cartilage) and hard tissue (bone) repair. This energy-rich molecule exhibits a property not shown by any other biopolymer: the delivery of metabolic energy, even extracellularly, necessary for the ATP-dependent tissue regeneration. This review summarizes the latest developments in nanobiomaterials based on these two evolutionarily old, regeneratively active materials, amorphous silica and amorphous polyP, highlighting their specific, partly unique properties and mode of action, and discussing their possible applications in human therapy. The results of initial proof-of-concept studies on patients demonstrating complete healing of chronic wounds are outlined. Keywords: biomaterials, nanoparticles, biosilica, polyphosphate, silicatein, regenerative medicine

Heinz C Schröder,1 Meik Neufurth,1 Huan Zhou,2 Shunfeng Wang,1 Xiaohong Wang,1 Werner E G Müller1 1ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; 2School of Health Sciences and Biomedical Engineering, Heibei University of Technology, Tianjin, People’s Republic of ChinaCorrespondence: Heinz C Schröder; Werner E G Müller, ERC Advanced Investigator Group, Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Duesbergweg 6, Mainz, 55128, Germany, Tel +49 6131 392 5791 ; +49 6131 392 5910, Email hschroed@uni-mainz.de; wmueller@uni-mainz.deAbstract: Inorganic polyphosphates (polyP) are long-chain polymers of orthophosphate residues, which, depending on the external conditions, can be present both physiologically and synthetically in either soluble, nanoparticulate or coacervate form. In recent years, these polymers have received increasing attention due to their unprecedented ability to exhibit both morphogenetic and metabolic energy delivering properties. There are no other physiological molecules that contain as many metabolically utilizable, high-energy bonds as polyP, making these polymers of particular medical interest as components of advanced hydrogel scaffold materials for potential applications in ATP-dependent tissue regeneration and repair. However, these polymers show physiological activity only in soluble form and in the coacervate phase, but not as stable metal-polyP nanoparticles. Therefore, understanding the mechanisms of formation of polyP coacervates and nanoparticles as well as their transformations is important for the design of novel materials for tissue implants, wound healing, and drug delivery and is discussed here.Keywords: polyphosphate nanoparticles, phase separation, biomaterial, metabolic energy, morphogenetic activity, tissue regeneration

Yongqiang Xiao,1,* Dayuan Xu,1,* Hongyuan Song,2,* Futing Shu,1 Pei Wei,3 Xiaolan Yang,3 Chenjian Zhong,3 Xiaohong Wang,4 Werner EG Müller,4 YongJun Zheng,1 Shichu Xiao,1,* Zhaofan Xia11Department of Burn Surgery, Changhai Hospital, Second Military Medical University, Shanghai, People’s Republic of China; 2Department of Ophthalmology, Changhai Hospital, Second Military Medical University, Shanghai 200433, People’s Republic of China; 3Department of Burns Surgery, Union Hospital, Fujian Medical University, Fuzhou 350001, People’s Republic of China; 4Erc Advanced Investigator Grant Research Group at the Institute for Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz 55128, Germany*These authors contributed equally to this workBackground: Less apoptosis and excessive growth of fibroblasts contribute to the progression of hypertrophic scar formation. Cuprous oxide nanoparticles (CONPs) could have not only inhibited tumor by inducing apoptosis and inhibiting proliferation of tumor cells, but also promoted wound healing. The objective of this study was to further explore the therapeutic effects of CONPs on hypertrophic scar formation in vivo and in vitro.Methods: In vivo, a rabbit ear scar model was established on New Zealand albino rabbits. Six full-thickness and circular wounds (10 mm diameter) were made to each ear. Following complete re-epithelization observed on postoperative day 14, an intralesional injection of CONPs or 5% glucose solution was conducted to the wounds. The photo and ultrasonography of each wound were taken every week and scars were harvested on day 35 for further histomorphometric analysis. In vitro, the role of CONPs in human hypertrophic scar fibroblasts (HSFs) apoptosis and proliferation were evaluated by Tunnel assay, Annexin V/PI staining, cell cycle analysis, and EdU proliferation assay. The endocytosis of CONPs by fibroblasts were detected through transmission electron microscopy (TEM) and the mitochondrial membrane potential and ROS production were also detected.Results: In vivo, intralesional injections of CONPs could significantly improve the scar appearance and collagen arrangement, and decreased scar elevation index (SEI). In vitro, CONPs could prominently inhibit proliferation and induce apoptosis in HSFs in a concentration-dependent manner. In addition, CONPs could be endocytosed into mitochondria，damage the mitochondrial membrane potential and increase ROS production.Conclusion: CONPs possessed the therapeutic potential in the treatment of hypertrophic scar by inhibiting HSFs proliferation and inducing HSFs apoptosis.Keywords: CONPs, hypertrophic scar, apoptosis, proliferation, mitochondria

This study reports the chemical investigation and cytotoxic activity of the secondary metabolites produced by the endophytic fungus Chaetomium sp. isolated from Salvia officinalis growing in Morocco. This plant was collected from the Beni-Mellal Mountain in Morocco and belongs to the Lamiaceae family and is named in Morocco “Salmia”. The endophytic fungus Chaetomium sp. was isolated from the tissues of the stem of this plant. The fungal strain was identified by PCR. The crude organic extract of the fungal strain was proven to be active when tested for cytotoxicity against L5178Y mouse lymphoma cells. Chemical investigation of the secondary metabolites showed that cochliodinol is the main component beside isocochliodinol. The structures of the isolated compounds were determined on the basis of NMR analysis (1H, 13C, COSY and HMBC) as well as by mass spectrometry using ESI (Electron Spray Ionisation) as source.

Abstract Introduction Maternal effects occur when the phenotype of the offspring is influenced by the phenotype of the mother, which in turn depends on her heritable state as well as on influences from the current and past environmental conditions. All of these pathways may, therefore, form significant sources of variation in maternal effects. Here, we focused on the maternal transfer of carotenoids and vitamin E to the egg yolk, using canaries as a model species. Maternal yolk carotenoids and vitamin E are known to generate significant phenotypic variation in offspring, representing examples of maternal effects. We studied the intra-individual consistency in deposition patterns across two years and the mother-daughter resemblance across two generations in order to estimate the level of heritable variation. The effects of the current environmental conditions were studied via a food supplementation experiment, while the consequences of past environmental conditions were estimated on the basis of the early growth trajectories. Results There was a significant effect of the current environmental conditions on the yolk carotenoid and vitamin E deposition, but this effect varied between antioxidant components. The deposition of yolk carotenoids and vitamin E were linked to the process of yolk formation. Past environmental conditions did not contribute to the variation in yolk carotenoid and vitamin E levels nor did we find significant heritable variation. Conclusions The transfer of carotenoids or vitamin E may be an example where current environmental variation is largely passed from the mother to the offspring, despite the numerous intermediate physiological steps that are involved. Differences in the effect of the environmental conditions as experienced by the mother during laying may be due to differences in availability as well as physiological processes such as competitive exclusion or selective absorption.

Abstract Background The purpose of our study was to analyze the clinical and radiological long-term outcomes of surgically treated traumatic knee dislocations and determine prognostic factors for outcome. Methods Retrospective consecutive series of patients treated surgically for traumatic knee dislocation with reconstruction/refixation of the anterior (ACL) and posterior cruciate ligaments (PCL) and primary complete repair of collaterals and posteromedial and posteromedial corner structures. 68 patients were evaluated clinically (IKDC score, SF36 health survey, Lysholm score, Knee Society score, Tegner score, visual analogue scale - VAS pain and satisfaction, Cooper test) and radiologically (weight bearing and stress radiographs) with a mean follow up of 12 ± 8 years. Instrumented anterior-posterior translation was measured (Rolimeter, KT-1000). Pearson correlation and stepwise regression analysis was used. Results 82% of patients (n = 56) returned to their previous work. At final follow-up 6 patients (9%) suffered from pain VAS > 3. The mean side-to-side difference of anterior/posterior translation (KT-1000, 134N) was 1.6 ± 1.6 mm and 2.6 ± 1.4 mm. Valgus and varus stress testing in 30° flexion was 40 days were significantly associated with worse outcome (p < 0.05). Conclusions Early complete reconstruction can achieve good functional results and patient satisfaction with overall restoration of sports and working capacity. Negative predictive factors for outcome were injury pattern, type of surgical procedure and timing of surgery.

Abstract Background Ethylene is a widely distributed alkene product which is formed enzymatically (e.g., in plants) or by photochemical reactions (e.g., in the upper oceanic layers from dissolved organic carbon). This gaseous compound was recently found to induce in cells from the marine sponge Suberites domuncula, an increase in intracellular Ca2+ level ([Ca2+]i) and an upregulation of the expression of two genes, the potential ethylene-responsive gene, SDERR, and a Ca2+/calmodulin-dependent protein kinase. Results Here we describe for the first time, that besides sponge cells, mammalian cell lines (mouse NIH-3T3 and human HeLa and SaOS-2 cells) respond to ethylene, generated by ethephon, with an immediate and strong, transient increase in [Ca2+]i level, as demonstrated using Fura-2 imaging method. A rise of [Ca2+]i level was also found following exposure to ethylene gas of cells kept under pressure (SaOS-2 cells). The upregulation of [Ca2+]i was associated with an increase in the level of the cell cycle-associated Ki-67 antigen. In addition, we show that the effect of ethephon addition to S. domuncula cells depends on the presence of calcium in the extracellular milieu. Conclusion The results presented in this paper indicate that ethylene, previously known to act as a mediator (hormone) in plants only, deserves also attention as a potential signaling molecule in higher vertebrates. Further studies are necessary to clarify the specificity and physiological significance of the effects induced by ethylene in mammalian cells.

Synaptic impairment may be the main cause of cognitive dysfunction in brain aging that is probably due to a reduction in synaptic contact between the axonal buttons and dendritic spines. Rho proteins including the small GTPase Rac1 have become key regulators of neuronal morphogenesis that supports synaptic plasticity. Small Rho- and Ras-GTPases are post-translationally modified by the isoprenoids geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), respectively. For all GTPases, anchoring in the plasma membrane is essential for their activation by guanine nucleotide exchange factors (GEFs). Rac1-specific GEFs include the protein T lymphoma invasion and metastasis 1 (Tiam1). Tiam1 interacts with the TrkB receptor to mediate the brain-derived neurotrophic factor (BDNF)-induced activation of Rac1, resulting in cytoskeletal rearrangement and changes in cellular morphology. The flavonoid 7,8-dihydroxyflavone (7,8-DHF) acts as a highly affine-selective TrkB receptor agonist and causes the dimerization and autophosphorylation of the TrkB receptor and thus the activation of downstream signaling pathways. In the current study, we investigated the effects of 7,8-DHF on cerebral lipid isoprenoid and Rho protein levels in male C57BL/6 mice aged 3 and 23 months. Aged mice were daily treated with 100 mg/kg b.w. 7,8-DHF by oral gavage for 21 days. FPP, GGPP, and cholesterol levels were determined in brain tissue. In the same tissue, the protein content of Tiam1 and TrkB in was measured. The cellular localization of the small Rho-GTPase Rac1 and small Rab-GTPase Rab3A was studied in total brain homogenates and membrane preparations. We report the novel finding that 7,8-DHF restored levels of the Rho proteins Rac1 and Rab3A in membrane preparations isolated from brains of treated aged mice. The selective TrkB agonist 7,8-DHF did not affect BDNF and TrkB levels, but restored Tiam1 levels that were found to be reduced in brains of aged mice. FPP, GGPP, and cholesterol levels were significantly elevated in brains of aged mice but not changed by 7,8-DHF treatment. Hence, 7,8-DHF may be useful as pharmacological tool to treat age-related cognitive dysfunction although the underlying mechanisms need to be elucidated in detail.

Background: Approved drugs for Alzheimer's disease (AD) only have a symptomatic effects and do not intervene causally in the course of the disease. Olesoxime (TRO19622) has been tested in AD disease models characterized by improved amyloid precursor protein processing (AβPP) and mitochondrial dysfunction., Methods: Three months old Thy-1-AβPP SL (tg) and wild type mice (wt) received TRO19622 (100 mg/kg b.w.) in supplemented food pellets for 15 weeks (tg TRO19622). Mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) levels were determined in dissociated brain cells (DBC). Respiration was analyzed in mitochondria isolated from brain tissue. Citrate synthase (CS) activity and beta-amyloid peptide (Aβ 1-40 ) levels were determined in brain tissue. Malondialdehyde (MDA) levels were determined as an indicator for lipid peroxidation. DBC and brain homogenates were additionally stressed with Rotenone and FeCl 2 , respectively. Mitochondrial respiration and Aβ 1-40 levels were also determined in HEK-AβPP sw -cells., Results: Treatment of mice did not affect the body weight. TRO19622 was absorbed after oral treatment (plasma levels: 6,2 μg/ml). Mitochondrial respiration was significantly reduced in brains of tg-mice. Subsequently, DBC isolated from brains of tg-mice showed significantly lower MMP but not ATP levels. TRO19622 increased the activity of respiratory chain complexes and reversed complex IV (CIV) activity and MMP. Moreover, DBC isolated from brains of tg TRO19622 mice were protected from Rotenone induced inhibition of complex I activity. TRO19622 also increased the respiratory activity in HEKsw-cells. MDA basal levels were significantly higher in brain homogenates isolated from tg-mice. TRO19622 treatment had no effects on lipid peroxidation. TRO19622 increased cholesterol levels but did not change membrane fluidity of synaptosomal plasma and mitochondrial membranes isolated from brain of mice. TRO19622 significantly increased levels of Aβ 1-40 in both, in brains of tg TRO19622 mice and in HEK sw cells., Conclusions: TRO19622 improves mitochondrial dysfunction but enhances Aβ levels in disease models of AD. Further studies must evaluate whether TRO19622 offers benefits at the mitochondrial level despite the increased formation of Aβ, which could be harmful., Competing Interests: Conflict of interest statement The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Dimebon (latrepirdine), an old antihistaminic drug, showed divergent results in two large clinical trials in Alzheimer disease (AD), which according to our review might be related to the specific pharmacological properties of the drug and the different patient populations included in both studies. Out of the many pharmacological effects of Dimebon, improvement of impaired mitochondrial function seeems to be most relevant for the substantial effects on cognition and behaviour reported in one of the studies, as these effects are already present at the low concentrations of dimebon measured in plasma and tissues of patients and experimental animals. Since impaired mitochondrial function seems to be the major driving force for the progression of the clinical symptoms and since most of the clinical benefits of dimebon originate from an effect on the symptomatic deterioration, mitochondrial improvement can also explain the lack of efficacy of this drug in another clinical trial where symptoms of the patiets remained stable for the time of the study. Accordingly, it seems worthwhile to reevaluate the clinical data to proof that clinical response is correlated with high levels of Neuropsychiatric Symptoms as these show a good relationship to the individual speed of symptomatic decline in AD patients related to mitochondrial dysfunction., Competing Interests: Conflict of Interest The authors declare no conflict of interest

Mitochondrial dysfunction plays an important role in brain aging and has emerged to be an early event in Alzheimer's disease (AD), contributing to neurodegeneration and the loss of physical abilities seen in patients suffering from this disease. We examined mitochondrial dysfunction in a cell culture model of AD (PC12APPsw cells) releasing very low amyloid-β (Aβ40) levels and thus mimicking early AD stages. Our data show that these cells have impaired energy metabolism, low ATP levels, and decreased endogenous mitochondrial respiration. Furthermore, protein levels of PGC1α as well as of Mitofusin 1 were decreased. PC12APPsw cells also showed increased mitochondrial content, probably due to an attempt to compensate the impaired mitochondrial function. Recent data showed that stabilized rice bran extract (RBE) protects from mitochondrial dysfunction in vivo Pharmacol Res. (2013) 76C, 17-27. To assess the effect of RBE on mitochondrial function, we treated PC12APPsw cells for 24 h with RBE. Key components of RBE are oryzanols, tocopherols, and tocotrienols, all substances that have been found to exert beneficial effects on mitochondrial function. RBE incubation elevated ATP production and respiratory rates as well as PGC1α protein levels in PC12APPsw cells, thus improving the impaired mitochondrial function assessed in our cell culture AD model. Therefore, RBE represents to be a promising nutraceutical for the prevention of AD.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the huntingtin gene (HTT). One prominent target of the mutant huntingtin protein (mhtt) is the mitochondrion, affecting its morphology, distribution, and function. Thus, mitochondria have been suggested as potential therapeutic targets for the treatment of HD. Olesoxime, a cholesterol-like compound, promotes motor neuron survival and neurite outgrowth in vitro, and its effects are presumed to occur via a direct interaction with mitochondrial membranes (MMs). We examined the properties of MMs isolated from cell and animal models of HD as well as the effects of olesoxime on MM fluidity and cholesterol levels. MMs isolated from brains of aged Hdh Q111/Q111 knock-in mice showed a significant decrease in 1,6-diphenyl-hexatriene (DPH) anisotropy, which is inversely correlated with membrane fluidity. Similar increases in MM fluidity were observed in striatal STHdh Q111/Q111 cells as well as in MMs isolated from brains of BACHD transgenic rats. Treatment of STHdh cells with olesoxime decreased the fluidity of isolated MMs. Decreased membrane fluidity was also measured in olesoxime-treated MMs isolated from brains of HD knock-in mice. In both models, treatment with olesoxime restored HD-specific changes in MMs. Accordingly, olesoxime significantly counteracted the mhtt-induced increase in MM fluidity of MMs isolated from brains of BACHD rats after 12 months of treatment in vivo, possibly by enhancing MM cholesterol levels. Thus, olesoxime may represent a novel pharmacological tool to treat mitochondrial dysfunction in HD.

Synaptic impairment rather than neuronal loss may be the leading cause of cognitive dysfunction in brain aging. Certain small Rho-GTPases are involved in synaptic plasticity, and their dysfunction is associated with brain aging and neurodegeneration. Rho-GTPases undergo prenylation by attachment of geranylgeranylpyrophosphate (GGPP) catalyzed by GGTase-I. We examined age-related changes in the abundance of Rho and Rab proteins in membrane and cytosolic fractions as well as of GGTase-I in brain tissue of 3- and 23-month-old C57BL/6 mice. We report a shift in the cellular localization of Rho-GTPases toward reduced levels of membrane-associated and enhanced cytosolic levels of those proteins in aged mouse brain as compared with younger mice. The age-related reduction in membrane-associated Rho proteins was associated with a reduction in GGTase-Iβ levels that regulates binding of GGPP to Rho-GTPases. Proteins prenylated by GGTase-II were not reduced in aged brain indicating a specific targeting of GGTase-I in the aged brain. Inhibition of GGTase-I in vitro modeled the effects of aging we observed in vivo. We demonstrate for the first time a decrease in membrane-associated Rho proteins in aged brain in association with down-regulation of GGTase-Iβ. This down-regulation could be one of the mechanisms causing age-related weakening of synaptic plasticity., (© 2014 International Society for Neurochemistry.)

Mitochondrial dysfunction plays a major role in the development of age-related neurodegenerative diseases and recent evidence suggests that food ingredients can improve mitochondrial function. In the current study we investigated the effects of feeding a stabilized rice bran extract (RBE) on mitochondrial function in the brain of guinea pigs. Key components of the rice bran are oryzanols, tocopherols and tocotrienols, which are supposed to have beneficial effects on mitochondrial function. Concentrations of α-tocotrienol and γ-carboxyethyl hydroxychroman (CEHC) but not γ-tocotrienol were significantly elevated in brains of RBE fed animals and thus may have provided protective properties. Overall respiration and mitochondrial coupling were significantly enhanced in isolated mitochondria, which suggests improved mitochondrial function in brains of RBE fed animals. Cells isolated from brains of RBE fed animals showed significantly higher mitochondrial membrane potential and ATP levels after sodium nitroprusside (SNP) challenge indicating resistance against mitochondrial dysfunction. Experimental evidence indicated increased mitochondrial mass in guinea pig brains, e.g. enhanced citrate synthase activity, increased cardiolipin as well as respiratory chain complex I and II and TIMM levels. In addition levels of Drp1 and fis1 were also increased in brains of guinea pigs fed RBE, indicating enhanced fission events. Thus, RBE represents a potential nutraceutical for the prevention of mitochondrial dysfunction and oxidative stress in brain aging and neurodegenerative diseases., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Statins have proven their effectiveness in the treatment of cardiovascular disease. This class of drugs has also attracted attention as a potential treatment for dissimilar diseases such as certain types of cancers and neurodegenerative diseases. What appears to be a contradiction is that, in the case of cancer, it has been suggested that statins increase apoptosis and alter levels of Bcl-2 family members (e.g., reduce Bcl-2 and increase Bax), whereas studies mainly using noncancerous cells report opposite effects. This review examined studies reporting on the effects of statins on Bcl-2 family members, apoptosis, cell death, and cell protection. Much, but not all, of the evidence supporting the pro-apoptotic effects of statins is based on data in cancer cell lines and the use of relatively high drug concentrations. Studies indicating an anti-apoptotic effect of statins are fewer in number and generally used much lower drug concentrations and normal cells. Those conclusions are not definitive, and certainly, there is a need for additional research to determine if statin repositioning is justified for noncardiovascular diseases.

Mitochondrial dysfunction represents a common early pathological event in brain aging and in neurodegenerative diseases, e.g., in Alzheimer's (AD), Parkinson's (PD), and Huntington's disease (HD), as well as in ischemic stroke. In vivo and ex vivo experiments using animal models of aging and AD, PD, and HD mainly showed improvement of mitochondrial function after treatment with polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA). Thereby, PUFA are particular beneficial in animals treated with mitochondria targeting toxins. However, DHA showed adverse effects in a transgenic PD mouse model and it is not clear if a diet high or low in PUFA might provide neuroprotective effects in PD. Post-treatment with PUFA revealed conflicting results in ischemic animal models, but intravenous administered DHA provided neuroprotective efficacy after acute occlusion of the middle cerebral artery. In summary, the majority of preclinical data indicate beneficial effects of n-3 PUFA in neurodegenerative diseases, whereas most controlled clinical trials did not meet the expectations. Because of the high half-life of DHA in the human brain clinical studies may have to be initiated much earlier and have to last much longer to be more efficacious., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Mitochondria are membrane bound organelles that provide cellular energy in form of ATP. In addition to ATP synthesis mitochondria are key regulators of calcium homeostasis, free radical production, steroid synthesis and apoptosis, each of these factors could also be associated with essential mechanisms involved in neurodegenerative diseases. Recent studies revealed that changes in mitochondria membrane fluidity might have a direct impact on membrane-based processes such as fission-associated morphogenic changes, opening of the mitochondrial permeability transition pore or oxidative phosphorylation at the complexes of the electron transport chain. We investigated synaptosomal plasma and mitochondrial membranes isolated from brains of mouse models for ageing, Alzheimer's disease, Huntington's disease and Amyotrophic lateral sclerosis. Membrane properties are disease specifically altered, identifying mitochondrial membranes as targets for possible therapeutic strategies in neurodegenerative diseases. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The global trend of the phenomenon of population ageing has dramatic consequences on public health and the incidence of neurodegenerative diseases. Physiological changes that occur during normal ageing of the brain may exacerbate and initiate pathological processes that may lead to neurodegenerative disorders, especially Alzheimer's disease (AD). Hence, the risk of AD rises exponentially with age. While there is no cure currently available, sufficient intake of certain micronutrients and secondary plant metabolites may prevent disease onset. Polyphenols are highly abundant in the human diet, and several experimental and epidemiological evidences indicate that these secondary plant products have beneficial effects on AD risks. This study reviews current knowledge on the potential of polyphenols and selected polyphenol-rich diets on memory and cognition in human subjects, focusing on recent data showing in vivo efficacy of polyphenols in preventing neurodegenerative events during brain ageing and in dementia. Concentrations of polyphenols in animal brains following oral administration have been consistently reported to be very low, thus eliciting controversial discussion on their neuroprotective effects and potential mechanisms. Whether polyphenols exert any direct antioxidant effects in the brain or rather act by evoking alterations in regulatory systems of the brain or even the body periphery is still unclear. To understand the mechanisms behind the protective abilities of polyphenol-rich foods, an overall understanding of the biotransformation of polyphenols and identification of the various metabolites arising in the human body is also urgently needed.

Increasing evidences suggest that mitochondrial dysfunction plays an important role in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD). Alterations of mitochondrial efficiency and function are mainly related to alterations in mitochondrial content, amount of respiratory enzymes, or changes in enzyme activities leading to oxidative stress, mitochondrial permeability transition pore opening, and enhanced apoptosis. More recently, structural changes of the network are related to bioenergetic function, and its consequences are a matter of intensive research. Several mitochondria-targeting compounds with potential efficacy in AD including dimebon, methylene blue, piracetam, simvastatin, Ginkgo biloba, curcumin, and omega-3 polyunsaturated fatty acids have been identified. The majority of preclinical data indicate beneficial effects, whereas most controlled clinical trials did not meet the expectations. Since mitochondrial dysfunction represents an early event in disease progression, one reason for the disappointing clinical results could be that pharmacological interventions might came too late. Thus, more studies are needed that focus on therapeutic strategies starting before severe disease progress.

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23 months) as compared to middle-aged mice (12 months) and young mice (3 months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.

Due to their role in producing energy, as major sources of free radicals, and as critical regulators of apoptosis, mitochondria play a dominant role in the central nervous system (CNS). Mitochondrial dysfunction represents one major pathomechanism of Alzheimer's disease (AD), including impaired function of mitochondrial respiratory chain complexes and deficits of mitochondrial dynamics, such as impaired balance between fission and fusion mechanisms and reduced mitochondrial trafficking. Major consequences are enhanced depletion of mitochondria in axons and dendrites, synaptic dysfunction, and finally neuronal loss. Interfering with impaired mitochondrial dynamics has been proposed as novel strategy for antidementia drugs. Dimebon has been shown to improve cognition in animal models and seems to be beneficial in AD patients. Regardless of the final proof of Dimebon's clinical efficacy, it might specifically interfere with mechanisms relevant for the cognitive decline, especially by improving impaired mitochondrial function and/or dynamics in AD. Herein, we tested the effects of Dimebon on mitochondrial function and dynamics in a cellular model, overexpressing neurotoxic Aβ peptides, one of the hallmarks of AD. Dimebon exerted pronounced effects on mitochondrial morphology, respiratory chain complex activities, and enlarged mitochondrial mass. In summary, form and function of mitochondria are altered in the Aβ overexpressing cell model and precisely those changes are restored by nanomolar Dimebon treatment. Our findings support the idea that Dimebon improves mitochondrial function and that these "disease specific" effects might be relevant for interpretation and planning of future clinical trials.

Siliceous spicules of the freshwater Baikal sponge Lubomirskia baicalensis contain several proteins including silicateins. Existences of four different genes of silicatein alpha (alpha1, alpha2, alpha3, alpha4) which are related to silicatein alpha from the sea sponges were found when cDNA library analysis was made. The intron-exon structure of the full-size silicatein alpha1 gene was determined. This gene has total length of 1988 bp and includes 6 introns (1007 bp) and 7 exons (981 bp). With use of mass-spectrometric analysis of the spicule proteins tryptic digest, two silicateins alpha were authentically found.

Cytosolic phospholipase A2 (cPLA2) preferentially liberates arachidonic acid (AA), which is known to be elevated in Alzheimer's disease (AD). The aim of this study was to investigate the possible relationship between enhanced nitric oxide (NO) generation observed in AD and cPLA2 protein level, phosphorylation, and AA release in rat pheochromocytoma cell lines (PC12) differing in amyloid beta secretion. PC12 control cells, PC12 cells bearing the Swedish double mutation in amyloid beta precursor protein (APPsw), and PC12 cells transfected with human APP (APPwt) were used. The transfected APPwt and APPsw PC12 cells showed an about 2.8- and 4.8-fold increase of amyloid beta (Abeta) secretion comparing to control PC12 cells. An increase of NO synthase activity, cGMP and free radical levels in APPsw and APPwt PC12 cells was observed. cPLA2 protein level was higher in APPsw and APPwt PC12 cells comparing to PC12 cells. Moreover, phosphorylated cPLA2 protein level and [3H]AA release were also higher in APP-transfected PC12 cells than in the control PC12 cells. An NO donor, sodium nitroprusside, stimulated [3H]AA release from prelabeled cells. The highest NO-induced AA release was observed in control PC12 cells, the effect in the other cell lines being statistically insignificant. Inhibition of cPLA2 by AACOCF3 significantly decreased the AA release. Inhibitors of nNOS and gamma-secretase reduced AA release in APPsw and APPwt PC12 cells. The basal cytosolic [Ca2+](i) and mitochondrial Ca2+ concentration was not changed in all investigated cell lines. Stimulation with thapsigargin increased the cytosolic and mitochondrial Ca2+ level, activated NOS and stimulated AA release in APP-transfected PC12 cells. These results indicate that Abeta peptides enhance the protein level and phosphorylation of cPLA2 and AA release by the NO signaling pathway.

Background: The lack convenient and inexpensive small animal model of HIV infection hampers significantly the research on immunopathogenesis, antiviral drugs, and vaccines., Objectives: Development of HIV model in small animals, i.e., cotton rats, was undertaken., Study Design: Cotton rats of both sexes were exposed to highly replicating HIV-1(zmb) strain by intraperitonial and retrobulbar routes. The infection was evaluated by morphological and clinical criteria and by amplification of HIV DNA integrated in animal tissues., Results: Six months after intraperitoneal infection the lymphoid tissues of all inoculated animals exhibited integrated proviral DNA. Despite viral integration no active systemic HIV replication was observed. Brain and spleen morphological changes indicated the presence of the inflammatory reaction, which was followed by a degenerative process. Clinical manifestations of the infection, while varied, revealed relative weight loss at three months after infection; apparent respiratory disorders; and death of some animals with signs of cachexia and alopecia., Conclusions: Clinical, morphological, and virology assessment of infected animals indicate that the virus crosses the interspecies barrier and cotton rats can be used as a small animal model of HIV infection.

Autosomal dominant forms of familial Alzheimer's disease (FAD) are caused by mutations of the amyloid precursor protein (APP) gene and by mutations of the genes encoding for presenilin 1 or presenilin 2. Simultaneously, evidence is provided that increased oxidative stress might play a crucial role in the rapid progression of the Swedish FAD. Here we investigated the effect of the Swedish double mutation (K670M/N671L) in the beta-amyloid precursor protein on oxidative stress-induced cell death mechanisms in PC12 cells. Western blot analysis and cleavage studies of caspase substrates revealed an elevated activity of the executor caspase 3 after treatment with hydrogen peroxide in cells containing the Swedish APP mutation. This elevated activity is the result of the enhanced activation of both intrinsic and extrinsic apoptosis pathways, including activation of caspase 2 and caspase 8. Furthermore, we observed an enhanced activation of JNK pathway and an attenuation of apoptosis by SP600125, a JNK inhibitor, through protection of mitochondrial dysfunction and reduction of caspase 9 activity. Our findings provide evidence that the massive neurodegeneration in early age of FAD patients could be a result of an increased vulnerability of neurons through activation of different apoptotic pathways as a consequence of elevated levels of oxidative stress.

Galectins are widely distributed sugar-binding proteins whose basic specificity for beta-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67-87]. By using this system, we quantitatively analyzed the interactions at 20 degrees C between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3-OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Le(x), Le(a)). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for alpha1-2Fuc-, alpha1-3Gal-, alpha1-3GalNAc-, or alpha2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either "branched", "repeated", or "substituted" glycans, while conserving their ability to recognize basic disaccharide units, Galbeta1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type.

Proteins featuring scavenger receptor cysteine-rich (SRCR) domains are prominent receptors known from vertebrates and from one phylum of invertebrates, the echinoderms. In the present study we report the first putative SRCR protein from the marine sponge Geodia cydonium (Porifera), a member of the lowest phylum of contemporary Metazoans. Two forms of SRCR molecules were characterized, which apparently represent alternative splicing of the same transcript. The long putative SRCR protein, of 1536 aa, features twelve SRCR repeats, a C-terminal transmembrane domain and a cytoplasmic tail. The sequence of the short form is identical with the long form except that it lacks a coding region near the C terminus, thus the 1195 aa deduced protein consists of only the first ten SRCR domains and the last 26 C-terminal aa residues, without the transmembrane domain. Homology searches revealed that the sponge putative SRCR protein shares with bovine T-cell antigen WC1 29.2% identity in 1054 aa overlap, 33.9% identity in 475 aa overlap with sea urchin speract and 56% identity in 110 aa overlap with macrophage scavenger receptor type I. Based upon the number and location of the conserved Cys residues, the sponge SRCR domain repeats were classified as belonging to group A of the SRCR superfamily. With twelve SRCR repeats, one more than those in any of the previously described SRCR proteins, and several membrane-bound and soluble forms, it seems that the most primitive known member of this family may be the structurally most complex one among SRCR containing proteins.

Human blood plasma, serum, peripheral blood mononuclear cells, and erythrocytes contain significant amounts of inorganic polyphosphates (ranging from 53 to 116 microM, in terms of phosphate residues). Here we demonstrate that at higher concentrations linear polyphosphates display cytoprotective and antiviral activity. Sodium tetrapolyphosphate and the longer polymers, with average chain lengths of 15, 34, and 91 phosphate residues, significantly inhibited human immunodeficiency virus type 1 (HIV-1) infection of cells in vitro at concentrations > or = 33.3 microg/ml (> or = 283-324 microM phosphate residues), whereas sodium tripolyphosphate was ineffective. In the tested concentration range, these compounds had no effect on cell growth. The longer-chain polyphosphates (polyphosphates with mean chain lengths of 15 and 34) but not sodium tripolyphosphate and sodium tetrapolyphosphate also inhibited HIV-1-induced syncytium formation at a concentration of 160 microg/ml (1.51-1.54 mM phosphate residues). The results obtained with the syncytium assay and by cell-virus binding experiments indicate that the anti-HIV effect of these nontoxic polyanions may be caused by binding of the compounds to both the host cell surface and the virus, thereby inhibiting adsorption of the virus. Competition experiments revealed that binding of [32P]polyphosphate to Molt-3 cells was only partially inhibited by the antibody OKT4A.

Ammonium salts of some anions decrease the potency of glycine in inhibiting (3H)strychnine binding associated with synaptic glycine receptors. A correspondence exists between the ability of the ammonium salts of anions to increase the IC50 of glycine in inhibiting the (3H) strychnine binding, their capacity to reduce the (3H) strychnine binding itself, and their capacity to reverse inhibitory postsynaptic potentials. The decrease of (3H)strychnine binding in the presence of chloride is abolished by sodium, while the decrease of the potency of glycine in inhibiting (3H)strychnine is not. Binding of (3H)strychnine is influenced by monovalent cations in a biphasic fashion. Concentrations of Li+, K+, and Na+ up to 150mM decrease (3H)strychnine binding, while higher concentrations of the cations increase (3H)strychnine binding. Inhibition by glycine of (3H)strychnine binding is enhanced by low concentrations of these cations.

The in vivo potencies of 4 beta-carboline derivatives as inhibitors of benzodiazepine receptor binding were investigated. For all 4 derivatives maximal inhibition was seen within 2 min after i.v. application but decreased continuously for the next 20 min and after this time accounted for less than one-third of maximal inhibition. The in vivo potencies of the beta-carbolines as inhibitors of benzodiazepine receptor binding are much smaller than one would expect from their affinities measured in vitro. Thus, it is assumed than only a small portion of the i.v. dose reaches the brain.