Metabolomics of the human aqueous humor

Introduction

The optical elements of the eye—cornea, lens, and vitreous humor—are avascular tissues, and their nutrition and waste removal are provided by aqueous humor (AH). The AH production occurs through the active secretion and the passive diffusion/ultrafiltration of blood plasma. The comparison of the metabolomic profiles of AH and plasma is important for understanding of the mechanisms of biochemical processes and metabolite transport taking place in vivo in ocular tissues.

Objectives

The work is aimed at the determination of concentrations of a wide range of most abundant metabolites in the human AH, the comparison of the metabolomic profiles of AH and serum, and the analysis of the post-mortem metabolomic changes in these two biological fluids.

Methods

The quantitative metabolomic profiling was carried out with the use of two independent methods—high-frequency ¹H NMR spectroscopy and HPLC with high-resolution ESI-MS detection.

Results

The concentrations of 71 most abundant metabolites in blood serum and AH from living patients and human cadavers have been measured. It has been found that the level of ascorbate in AH is by two orders of magnitude higher than that in serum; the levels of other metabolites are either similar to that in serum, or differ from that by a factor of 2–5. The post-mortem metabolomic composition of both serum and AH undergoes rapid and strong changes.

Conclusion

The differences between the metabolomic profiles of AH and serum for majority of metabolites can be attributed to the metabolic activity of the ocular tissues leading to the lack or excess of some metabolites, while the high concentration of ascorbate in AH demonstrates the activity of ascorbate-specific pumps at the blood-aqueous border. The post-mortem metabolomic changes are caused by the disruption of the major biochemical cycles and cell lysis. These changes should be taken into account in the analysis of disease-induced changes in post-mortem samples of the ocular tissues.

     

Redox-dependent mechanisms of regulation of breast epithelial cell proliferation

Activation of free radical oxidation in various types of cells, including breast epithelial cells, can lead to damage to macromolecules, particularly proteins involved in regulation of proliferation and programmed cell death. The glutathione, glutaredoxin and thioredoxin systems play an essential role in maintaining intracellular redox homeostasis. In this regard, modulation of the redox status of cells by means of a blocker and a protector of SH-groups of proteins can be used as a model for studying the role of redox proteins and glutathione in regulation of cell proliferation during the development of various pathological processes. In this study the state of thioredoxin, glutaredoxin, glutathione systems and their role in the regulation of HBL-100 breast epithelial cell proliferation during modulation of the redox status by using N-ethylmaleimide (NEM) and 1,4-dithioerythritol (DTE) have been investigated. The modulation of the redox status of the breast epithelial cells by the blocker (NEM) and the protector (DTE) of thiol groups of proteins and peptides influenced the functional activity of glutathione-dependent enzymes, glutaredoxin, thioredoxin, and thioredoxin reductase by changing concentrations of GSH and GSSG. Modulation of the redox status of HBL-100 cells was accompanied by an increase in the number of cells in the S phase of the cell cycle and a decrease of cells in G₀/G₁ and G₂/M phases as compared with the intact cell culture. The proposed method for evaluating the proliferative activity of cells during modulation of their redox state can be used in the development of new therapeutic approaches for treatment of diseases accompanied by the development of oxidative stress.     

Anaerobic Microbial Degradation of Polypropylene and Polyvinyl Chloride Samples

Microbiology - Resistance to biodegradation, which is among the most advantageous features of synthetic polymers, is also the reason for their accumulation in the environment and therefore...     

Elevated Temperatures Inhibit Ferredoxin-Dependent Cyclic Electron Flow around Photosystem I

In experiments with barley (Hordeum vulgare L.) leaves, absorbance changes at 830 nm induced by far-red light were measured as indicator of redox conversions of primary electron donor (P700) of photosystem I (PSI). Using this method, the action of elevated temperature (45°C, 5 min) on PSI-driven electron transport through alternative pathways was examined. Thermally induced inactivation was found to transform nonmonotonic photooxidation of P700, induced by far-red light in untreated leaves, into a fast and monotonic process completed within 1-s illumination. The short-term heating of leaves fully eliminated the fast component in the kinetics of P700⁺ dark reduction, related to operation of ferredoxin-dependent cyclic electron transport around PSI. At the same time, thermoinactivation substantially accelerated the slow and middle components of dark P700⁺ reduction, i.e., the components determined by arrival of electrons to PSI from reductants located in the chloroplast stroma. The latter effect was also observed after heating of leaves pretreated with antimycin A or methyl viologen; both agents are known to inhibit the ferredoxin-dependent electron transport. It is concluded that the heat treatment of leaves inhibits the ferredoxin-dependent pathway of electron transport around PSI and activates electron transport through alternative routes providing reducing equivalents to PSI from stromal reductants.     

Interaction of exogenous quinones with membranes of higher plant chloroplasts: modulation of quinone capacities as photochemical and non-photochemical quenchers of energy in Photosystem II during light-dark transitions

Light modulation of the ability of three artificial quinones, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichloro-p-benzoquinone (DCBQ), and tetramethyl-p-benzoquinone (duroquinone), to quench chlorophyll (Chl) fluorescence photochemically or non-photochemically was studied to simulate the functions of endogenous plastoquinones during the thermal phase of fast Chl fluorescence induction kinetics. DBMIB was found to suppress by severalfold the basal level of Chl fluorescence (F(o)) and to markedly retard the light-induced rise of variable fluorescence (F(v)). After irradiation with actinic light, Chl fluorescence rapidly dropped down to the level corresponding to F(o) level in untreated thylakoids and then slowly declined to the initial level. DBMIB was found to be an efficient photochemical quencher of energy in Photosystem II (PSII) in the dark, but not after prolonged irradiation. Those events were owing to DBMIB reduction under light and its oxidation in the dark. At high concentrations, DCBQ exhibited quenching behaviours similar to those of DBMIB. In contrast, duroquinone demonstrated the ability to quench F(v) at low concentration, while F(o) was declined only at high concentrations of this artificial quinone. Unlike for DBMIB and DCBQ, quenched F(o) level was attained rapidly after actinic light had been turned off in the presence of high duroquinone concentrations. That finding evidenced that the capacity of duroquinone to non-photochemically quench excitation energy in PSII was maintained during irradiation, which is likely owing to the rapid electron transfer from duroquinol to Photosystem I (PSI). It was suggested that DBMIB and DCBQ at high concentration, on the one hand, and duroquinone, on the other hand, mimic the properties of plastoquinones as photochemical and non-photochemical quenchers of energy in PSII under different conditions. The first model corresponds to the conditions under which the plastoquinone pool can be largely reduced (weak electron release from PSII to PSI compared to PSII-driven electron flow from water under strong light and weak PSI photochemical capacity because of inactive electron transport on its reducing side), while the second one mimics the behaviour of the plastoquinone pool when it cannot be filled up with electrons (weak or moderate light and high photochemical competence of PSI).     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.