Aleksandar Bajić

Protective role of fructose in the metabolism of astroglial C6 cells exposed to hydrogen peroxide.

Astroglial cells represent the main line of defence against oxidative damage related to neurodegeneration. Therefore, protection of astroglia from an excess of reactive oxygen species could represent an important target of the treatment of such conditions. The aim of our study was to compare the abilities of glucose and fructose, the two monosaccharides used in diet and infusion, to protect C6 cells from hydrogen peroxide (H(2)O(2))-mediated oxidative stress. It was observed using confocal microscopy with fluorescent labels and the MTT test that fructose prevents changes of oxidative status of the cells exposed to H(2)O(2) and preserves their viability. Even more pronounced protective effects were observed for fructose 1,6-bis(phosphate). We propose that fructose and its intracellular forms prevent H(2)O(2) from participating in the Fenton reaction via iron sequestration. As fructose and fructose 1,6-bis(phosphate) are able to pass the blood-brain barrier, they could provide antioxidative protection of nervous tissue in vivo. So, in contrast to the well-known negative effects of frequent consumption of fructose under physiological conditions, acute infusion or ingestion of fructose or fructose 1,6-bis(phosphate) could be of benefit in the cytoprotective therapy of neurodegenerative disorders related to oxidative stress.

Relevance of the ability of fructose 1,6-bis(phosphate) to sequester ferrous but not ferric ions

The cytoprotective activity of F16BP has been documented in severe conditions such as convulsions, reperfusion injury, septic shock, diabetic complications, hypothermia-induced injury, UV-provoked skin damage and in other processes including apoptosis and excitotoxicity. F16BP shows very efficient cytoprotective activity in astroglial cells exposed to H(2)O(2)-provoked oxidative stress and during neuronal injury caused by hypoxic conditions. As most of the aforementioned processes involve iron activity-related conditions, we investigated the ferric and ferrous iron binding properties of F16BP under physiological conditions using (31)P NMR and EPR spectroscopy. Our results indicate that cytoprotective F16BP activity is predominantly based on ferrous iron sequestration. (31)P NMR spectroscopy of F16BP employing paramagnetic properties of iron clearly showed that F16BP forms stabile complexes with Fe(2+) which was verified by EPR of another divalent cation-Mn(2+). On the other hand, F16BP does not sequester ferric iron nor does it increase its redox activity as shown by (31)P NMR and EPR spin-trapping. Therefore, F16BP may be beneficial in neurodegenerative and other conditions that are characterised by ferric iron stores and deposits.

Fluctuating vs. Continuous Exposure to H2O2: The Effects on Mitochondrial Membrane Potential, Intracellular Calcium, and NF-κB in Astroglia

The effects of H2O2 are widely studied in cell cultures and other in vitro systems. However, such investigations are performed with the assumption that H2O2 concentration is constant, which may not properly reflect in vivo settings, particularly in redox-turbulent microenvironments such as mitochondria. Here we introduced and tested a novel concept of fluctuating oxidative stress. We treated C6 astroglial cells and primary astrocytes with H2O2, using three regimes of exposure – continuous, as well as fluctuating at low or high rate, and evaluated mitochondrial membrane potential and other parameters of mitochondrial activity – respiration, reducing capacity, and superoxide production, as well as intracellular ATP, intracellular calcium, and NF-κB activation. When compared to continuous exposure, fluctuating H2O2 induced a pronounced hyperpolarization in mitochondria, whereas the activity of electron transport chain appears not to be significantly affected. H2O2 provoked a decrease of ATP level and an increase of intracellular calcium concentration, independently of the regime of treatment. However, fluctuating H2O2 induced a specific pattern of large-amplitude fluctuations of calcium concentration. An impact on NF-κB activation was observed for high rate fluctuations, whereas continuous and low rate fluctuating oxidative stress did not provoke significant effects. Presented results outline the (patho)physiological relevance of redox fluctuations.

Actinidin, a protease from kiwifruit, induces changes in morphology and adhesion of T84 intestinal epithelial cells

Actinidin belongs to the papain-like family of cysteine proteases and is a major kiwifruit allergen. In this study, the effect of actinidin on cellular morphology and adhesion of T84 intestinal cells was investigated. Both rounding and detachment of T84 cells were observed upon actinidin treatment. The morphological changes and cell desquamation was protease-dependent, as well as time- and concentration-dependent. Changes of intercellular adhesion and adhesion of epithelial cells to collagen upon actinidin treatment could be responsible for the cell rounding and give rise to discontinuous breaches in the epithelial monolayer observed in this study. Actinidins action on cell morphology, adhesion and monolayer integrity were not due to compromised viability of T84 epithelial cells, as confirmed by MTT assay and flow cytometric analysis of the cell cycle. Damage to the epithelial monolayer of the intestine induced by actinidin should be further evaluated as an important factor in the development of kiwifruit allergy and other intestinal disorders.

Short-Term Facilitation and Depression in the Cerebellum: Some Observations on Wild-Type and Mutant Rodents Deficient in the Extracellular Matrix Molecule Tenascin C

Abstract: Short‐term plasticity was studied on synapses to Purkinje cells (PC): paired‐pulse facilitation in parallel fibers (PF) and paired‐pulse depression in climbing fibers (CF). Both phenomena relate to synaptic strength. These forms of short‐term plasticity were tested on cerebellar slices in rat by early postnatal synchronous stimulation of olivary neurons (i.e. CFs) with harmaline and by inhibition of a metabotropic glutamate receptor (mGluR) as well as in mice that were deficient in the extracellular matrix glycoprotein tenascin‐C. Harmaline stimulation delayed the developmental competition between CF inputs and maintained multiple innervation. Paired‐pulse depression of the CF‐PC synapse after harmaline treatment was more expressed. However, paired‐pulse facilitation in PF‐PC synapses remained unchanged. Electrophysiological responses of postsynaptic mGluR1 in CF‐PC synapses could be obtained only with AMPA receptors blocked and glutamate uptake impaired. The mGluR1‐specific antagonist CPCCOEt suppressed the CF‐mGluR EPSC in some PCs and potentiated it in other PCs. CF paired‐pulse depression was not changed with CPCCOEt, thus excluding a presynaptic effect. The postsynaptic effect was underlined by CPCCOEt‐induced rise in amplitude of EPSC and by a prolongation of its decay time. Tenascins are extracellular matrix glycoproteins that may restrict the regenerative capacity of the nervous tissue. Testing short‐term presynaptic plasticity in tenascin‐C‐deficient mice showed that CF paired‐pulse depression was less expressed while PF paired‐pulse facilitation was augmented except in a group of cells where there was even depression. The results underline differences in forms of short‐term plasticity with regard to susceptibility to diverse modulatory factors.

Expression of Ecto-Nucleoside Triphosphate Diphosphohydrolase1-3 (NTPDase1-3) by Cortical Astrocytes After Exposure to Pro-inflammatory Factors In Vitro

Nucleoside triphosphate diphosphohydrolases (NTPDases) are ecto-enzymes catalyzing the first step of sequential hydrolysis of extracellular ATP to adenosine, as the final product. Among eight members of NTPDase family, NTPDases1–3 have been shown to be expressed in the brain. Although altered NTPDase expression has been observed in relation to cell death and reactive gliosis in several experimentally induced neuropathologies, regulators of NTPDases expression and function are largely unknown. The present study explored the effects of several inflammatory factors (i.e., INF-γ, TNF-α, LPS, peroxide, and glutamate) on NTPDase1–3 activity and expression by cultured cortical astrocytes. We were able to demonstrate that INF-γ and TNF-α increased both ATP and ADP hydrolysis, while LPS specifically increased ATP hydrolysis. Consistent with the observed enhanced nucleotidase activity, INF-γ induced the upregulation of NTPDase1 at the mRNA and protein level. Furthermore, we were able to demonstrate that INF-γ and TNF-α decreased the relative abundance of dominant astrocytic NTPDase2 in favor of NTPDase1. In summary, these results suggest that INF-γ, TNF-α, and LPS may be relevant in vivo regulators of NTPDase expression in neuropathologies associated with neuroinflammation.

The effect of kiwifruit (Actinidia deliciosa) cysteine protease actinidin on the occludin tight junction network in T84 intestinal epithelial cells.

Actinidin, a kiwifruit cysteine protease, is a marker allergen for genuine sensitization to this food allergen source. Inhalatory cysteine proteases have the capacity for disruption of tight junctions (TJs) enhancing the permeability of the bronchial epithelium. No such properties have been reported for allergenic food proteases so far. The aim was to determine the effect of actinidin on the integrity of T84 monolayers by evaluating its action on the TJ protein occludin. Immunoblot and immunofluorescence were employed for the detection of occludin protein alterations. Gene expression was evaluated by RT-PCR. Breach of occludin network was assessed by measuring transepithelial resistance, blue dextran leakage and passage of allergens from the apical to basolateral compartment. Actinidin exerted direct proteolytic cleavage of occludin; no alteration of occludin gene expression was detected. There was a reduction of occludin staining upon actinidin treatment as a consequence of its degradation and dispersion within the membrane. There was an increase in permeability of the T84 monolayer resulting in reduced transepithelial resistance, blue dextran leakage and passage of allergens actinidin and thaumatin-like protein from the apical to basolateral compartment. Opening of TJs by actinidin may increase intestinal permeability and contribute to the process of sensitization in kiwifruit allergy.

Spectral sensitivity of the electroretinogram b-wave in dark-adapted Prussian carp ( Carassius gibelio Bloch, 1782)

One of the purposes of this study was to examine whether b-wave measurements can be used in the evaluation of scotopic spectral sensitivity in Prussian carp measurements when the eyes were surgically deprived of cornea, lens, and most of the vitreous. Another goal was testing the new fitting procedure for A2-based photopigments. Using fitted amplitude-log intensity functions for threshold calculation, and two models for computer-assisted fitting of spectral sensitivity curves, no significant differences in λmax were found between rod photopigments and b-wave-based spectral sensitivity.

Metabotropic Glutamate Receptor-Mediated Currents at the Climbing Fiber to Purkinje Cell Synapse

Different forms of synaptic plasticity in the cerebellum expressed at the synapses onto Purkinje cells (PCs) are mediated by membrane metabotropic glutamate receptors (mGluRs). There are three main mGluR groups with a total of 8 subtypes. Although mGluRs are also found at the climbing fiber (CF) to PC synapses, the distribution and biological activity of their types is not well-known. Using whole cell patch-clamp recordings from PCs in rat cerebellar slices with inhibitors of ionotropic receptors and glutamate uptake blockers we demonstrate a complex pharmacology of currents obtained by CF stimulation. The mGluR1 specific antagonist CPCCOEt in a group of cells suppressed this response, but in a similar number of other cells it induced a potentiating effect. It was found that a switch between these two biopharmaceutical effects might occur with age.

Influence of photic environment on the form of the fish electroretinographic off-response.

Abstract: Scotopic electroretinogram of dogfish shark (Scylliorhinus canicula) and eel (Anguilla anguilla) is characterized by a negative off‐response, changing in sign under photopic condition. It increased under the effect of increased background illumination, but its amplitude never exceeded that of the b‐wave. On the other hand, dark‐adapted electroretinograms of two perch‐like species, perch (Perca fluviatilis) and painted comber (Serranus scriba), exhibited a positive off‐wave, exceeding the b‐wave amplitude under bright photopic conditions.