Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways

The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HT's strengthening of the antiapoptotic actions of the endogenous antioxidant system.     

Ecophysiological analysis of moss-dominated biological soil crusts and their separate components from the Succulent Karoo, South Africa

Biological soil crusts, formed by an association of soil particles with cyanobacteria, lichens, mosses, fungi and bacteria in varying proportions, live in or directly on top of the uppermost soil layer. To evaluate their role in the global carbon cycle, gas exchange measurements were conducted under controlled conditions. Moss-dominated soil crusts were first analyzed as moss tufts on soil, then the mosses were removed and the soil was analyzed separately to obtain the physiological response of both soil and individual moss stems. Net photosynthetic response of moss stems and complete crusts was decreased by insufficient and excess amounts of water, resulting in optimum curves with similar ranges of optimum water content. Light saturation of both sample types occurred at high irradiance, but moss stems reached light compensation and saturation points at lower values. Optimum temperatures of moss stems ranged between 22 and 27°C, whereas complete crusts reached similar net photosynthesis between 7 and 27°C. Under optimum conditions, moss stems reached higher net photosynthesis (4.0 vs. 2.8 μmol m^?2 s^?1) and lower dark respiration rates (?0.9 vs. ?2.4 μmol m^?2 s^?1). Respiration rates of soil without moss stems were high (up to ?2.0 μmol m^?2 s^?1) causing by far lower absolute values of NP/DR ratios of soil crusts as compared to moss stems. In carbon balances, it therefore has to be clearly distinguished between measurements of soil crust components versus complete crusts. High rates of soil respiration may be caused by leaching of mosses, creating high-nutrient microsites that favor microorganism growth.     

Nongenomic effects of thyroid hormones on the immune system cells: New targets, old players

It is now widely accepted that thyroid hormones, l-thyroxine (T(4)) and 3,3',5-triiodo-l-thyronine (T(3)), act as modulators of the immune response. Immune functions such as chemotaxis, phagocytosis, generation of reactive oxygen species, and cytokine synthesis and release, are altered in hypo- and hyper-thyroid conditions, even though for many immune cells no clear correlation has been found between altered levels of T(3) or T(4) and effects on the immune responses. Integrins are extracellular matrix proteins that are important modulators of many cellular responses, and the integrin αvβ3 has been identified as a cell surface receptor for thyroid hormones. Rapid signaling via this plasma membrane binding site appears to be responsible for many nongenomic effects of thyroid hormones, independent of the classic nuclear receptors. Through the integrin αvβ3 receptor the hormone can activate both the ERK1/2 and phosphatidylinositol 3-kinase pathways, with downstream effects including intracellular protein trafficking, angiogenesis and tumor cell proliferation. It has recently become clear that an important downstream target of the thyroid hormone nongenomic pathway may be the mammalian target of rapamycin, mTOR. New results demonstrate the capability of T(3) or T(4) to induce in the short time range important responses related to the immune function, such as reactive oxygen species production and cell migration in THP-1 monocytes. Thus thyroid hormones seem to be able to modulate the immune system by a combination of rapid nongenomic responses interacting with the classical nuclear response.     

Critical differences in HIV-1 and HIV-2 protease specificity for clinical inhibitors

Clinical inhibitor amprenavir (APV) is less effective on HIV‐2 protease (PR₂) than on HIV‐1 protease (PR₁). We solved the crystal structure of PR₂ with APV at 1.5 Å resolution to identify structural changes associated with the lowered inhibition. Furthermore, we analyzed the PR₁ mutant (PR_1M) with substitutions V32I, I47V, and V82I that mimic the inhibitor binding site of PR₂. PR_1M more closely resembled PR₂ than PR₁ in catalytic efficiency on four substrate peptides and inhibition by APV, whereas few differences were seen for two other substrates and inhibition by saquinavir (SQV) and darunavir (DRV). High resolution crystal structures of PR_1M with APV, DRV, and SQV were compared with available PR₁ and PR₂ complexes. Val/Ile32 and Ile/Val47 showed compensating interactions with SQV in PR_1M and PR₁, however, Ile82 interacted with a second SQV bound in an extension of the active site cavity of PR_1M. Residues 32 and 82 maintained similar interactions with DRV and APV in all the enzymes, whereas Val47 and Ile47 had opposing effects in the two subunits. Significantly diminished interactions were seen for the aniline of APV bound in PR_1M and PR₂ relative to the strong hydrogen bonds observed in PR₁, consistent with 15‐ and 19‐fold weaker inhibition, respectively. Overall, PR_1M partially replicates the specificity of PR₂ and gives insight into drug resistant mutations at residues 32, 47, and 82. Moreover, this analysis provides a structural explanation for the weaker antiviral effects of APV on HIV‐2.     

Gambling on putative biomarkers of osteoarthritis and osteochondrosis by equine synovial fluid proteomics

Osteoarthritis (OA) and osteochondrosis (OC) are two of the main challenges in orthopedics, whose definitive diagnosis is usually based on radiographic/arthroscopic evidences. Their early diagnosis should allow preventive or timely therapeutic actions, which are generally precluded from the poor relationships occurring between symptomatologic and radiographic evidences. These limitations should be overcome by improving the knowledge on articular tissue metabolism and on molecular factors regulating its normal homeostasis, also identifying novel OA and OC biomarkers suitable for their earlier diagnoses, whenever clinical/pathological inflammatory scenarios between these joint diseases seem somewhat related. To identify proteins involved in their aetiology and progression, we undertook a differential proteomic analysis of equine synovial fluid (SF), which compared the protein pattern of OA or OC patients with that of healthy individuals. Deregulated proteins in OA and OC included components related to inflammatory state, coagulation pathways, oxidative stress and matrix damage, which were suggestive of pathological alterations in articular homeostasis, plasma-SF exchange, joint nutritional status and vessel permeability. Some proteins seemed commonly deregulated in both pathologies indicating that, regardless of the stimulus, common pathways are affected and/or the animal joint uses the same molecular mechanisms to restore its homeostasis. On the other hand, the increased number of deregulated proteins observed in OA with respect to OC, together with their nature, confirmed the high inflammatory character of this disease. Some deregulated proteins in OA found a verification by analyzing the SF of injured arthritic joints following autologous conditioned serum treatment, an emergent therapy that provides positive results for both human and equine OA. Being the horse involved in occupational/sporting activities and considered as an excellent animal model for human joint diseases, our data provide suggestive information for tentative biomedical extrapolations, allowing to overcome the limitations in joint size and workload that are typical of other small animal models.     

Proteomics of rat biological fluids--the tenth anniversary update

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.     

Plasma protein changes in horse after prolonged physical exercise: a proteomic study

Physical exercise induces various stress responses and metabolic adaptations that have not yet been completely elucidated. Novel biomarkers are needed in sport veterinary medicine to monitor training levels and to detect subclinical conditions that can develop into exercise-related diseases. In this study, protein modifications in horse plasma induced by prolonged, aerobic physical exercise were investigated by using a proteomic approach based on 2-DE and combined mass spectrometry procedures. Thirty-eight protein spots, associated with expression products of 13 genes, showed significant quantitative changes; spots identified as membrane Cu amine oxidase, α-1 antitrypsin, α-1 antitrypsin-related protein, caeruloplasmin, α-2 macroglobulin and complement factor C4 were augmented in relative abundance after the race, while haptoglobin β chain, apolipoprotein A-I, transthyretin, retinol binding protein 4, fibrinogen γ chain, complement factor B and albumin fragments were reduced. These results indicate that prolonged physical exercise affects plasma proteins involved in pathways related to inflammation, coagulation, immune modulation, oxidant/antioxidant activity and cellular and vascular damage, with consequent effects on whole horse metabolism.