Ferenc Gallyas

High-Grade Intensification of the End-Product of the Diaminobenzidine Reaction for Peroxidase Histochemistry

A simple and reliable method is described for the intensification of the end-product of the diaminobenzidine reaction demonstrating peroxidase activity. After completing the diaminobenzidine reaction, the preparations to be intensified are immersed first in thioglycolic acid solution, then in distilled water, and finally in a special physical developer containing silver nitrate.

Direct effect of Taxol on free radical formation and mitochondrial permeability transition.

To elucidate the potential role of mitochondria in Taxol-induced cytotoxicity, we studied its direct mitochondrial effects. In Percoll-gradient purified liver mitochondria, Taxol induced large amplitude swelling in a concentration-dependent manner in the microM range. Opening of the permeability pore was also confirmed by the access of mitochondrial matrix enzymes for membrane impermeable substrates in Taxol-treated mitochondria. Taxol induced the dissipation of mitochondrial membrane potential (DeltaPsi) determined by Rhodamine123 release and induced the release of cytochrome c from the intermembrane space. All these effects were inhibited by 2.5 microM cyclosporine A. Taxol significantly increased the formation of reactive oxygen species (ROS) in both the aqueous and the lipid phase as determined by dihydrorhodamine123 and resorufin derivative. Cytochrome oxidase inhibitor CN(-), azide, and NO abrogated the Taxol-induced mitochondrial ROS formation while inhibitors of the other respiratory complexes and cyclosporine A had no effect. We confirmed that the Taxol-induced collapse of DeltaPsi and the induction of ROS production occurs in BRL-3A cells. In conclusion, Taxol-induced adenine nucleotide translocase-cyclophilin complex mediated permeability transition, and cytochrome oxidase mediated ROS production. Because both cytochrome c release and mitochondrial ROS production can induce suicide pathways, the direct mitochondrial effects of Taxol may contribute to its cytotoxicity.

Novel phenanthridinone inhibitors of poly(adenosine 5'-diphosphate-ribose) synthetase: Potent cytoprotective and antishock agents

ObjectiveTo synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5′-diphosphate [ADP]-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock. DesignRandomized, prospective laboratory study. SettingResearch laboratory. SubjectsMurine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats. InterventionsMacrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis. Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity. Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1–10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor. The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability. Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally). PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment. Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured. In addition, survival rates and vascular contractile and relaxant responses were recorded. Measurements and Main ResultsAppropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents. The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM. PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34. The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized). The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages. PJ34 was selected for the subsequent in vivo studies. PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse. PJ34 reduced peak plasma concentrations of tumor necrosis factor-&agr;, interleukin-1&bgr;, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats. PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut. Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34. Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment). ConclusionsThe novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

Golgi-like demonstration of “dark” neurons with an argyrophil III method for experimental neuropathology

SummaryA silver method is proposed for the selective, well-contrasted and reproducible demonstration of “dark” neurons in frozen, vibratome and paraffin sections cut at a thickness of 5 to 200 μm from aldehyde-fixed brains. The Golgi-like staining of the dendrites enables asorting of “dark” neurons according to characteristic neuron classifications. The staining procedure includes an esterification with 1-propanol, a treatment with diluted acetic acid and development. The esterification strongly increases the argyrophilia of both “dark” neurons and mitochondria. Unwanted co-staining of mitochondria is suppressed by the acetic acid treatment, while a special developer is used to render the staining controllable. The applicability of the method to experimental neuropathology is demonstrated by Golgi-like staining of “dark” neurons in rat brains exposed, before transcardial perfusion-fixation and delayed autopsy, to various pathological conditions including ischemia, hypoglycemia, trauma, status epilepticus, deafferentation and poisoning with kainic acid, colchicine and sodium azide, respectively.

Decrease of the inflammatory response and induction of the Akt/protein kinase B pathway by poly-(ADP-ribose) polymerase 1 inhibitor in endotoxin-induced septic shock

The lack of efficacy of anti-inflammatory drugs, anti-coagulants, anti-oxidants, etc. in critically ill patients has shifted interest towards developing alternative treatments. Since inhibitors of the nuclear enzyme poly-(ADP-ribose) polymerase (PARP) were found to be beneficial in many pathophysiological conditions associated with oxidative stress and PARP-1 knock-out mice proved to be resistant to bacterial lipopolysaccharide (LPS)-induced septic shock, PARP inhibitors are candidates for such a role. In this study, the mechanism of the protective effect of a potent PARP-1 inhibitor, PJ34 was studied in LPS-induced (20mg/kg, i.p.) septic shock in mice. We demonstrated a significant inflammatory response by magnetic resonance imaging in the dorsal subcutaneous region, in the abdominal regions around the kidneys and in the inter-intestinal cavities. We have found necrotic and apoptotic histological changes as well as obstructed blood vessels in the liver and small intestine. Additionally, we have detected elevated tumor necrosis factor-alpha levels in the serum and nuclear factor kappa B activation in liver of LPS-treated mice. Pre-treating the animals with PJ34 (10mg/kg, i.p.), before the LPS challenge, besides rescuing the animals from LPS-induced death, attenuated all these changes presumably by activating the phosphatidylinositol 3-kinase-Akt/protein kinase B cytoprotective pathway.

A highly sensitive one-step method for silver intensification of the nickel-diaminobenzidine endproduct of peroxidase reaction.

We have developed a new technique which makes silver intensification of the oxidatively polymerized diaminobenzidine (DAB), the endproduct of peroxidase reaction, less laborious without any loss in selectivity or sensitivity. The new technique is based on two strategies: (a) increasing the argyrophilia of the DAB by modifying its polymerization with Ni ions, and (b) decreasing tissue argyrophila by using a mildly acidic physical developer instead of the alkaline one previously presented. Because the nickel modification takes place in the DAB substrate solution, i.e., in the final step of the peroxidase reaction, only one additional step, the physical development, must be carried out if intensification is needed.

The existence of biexponential signal decay in magnetic resonance diffusion-weighted imaging appears to be independent of compartmentalization.

It is generally believed that the apparent diffusion coefficient (ADC) changes measured by diffusion‐weighted imaging (DWI) in brain pathologies are related to alterations in the water compartments. The aim of this study was to elucidate the role of compartmentalization in DWI via biexponential analysis of the signal decay due to diffusion. DWI experiments were performed on mouse brain over an extended range of b‐values (up to 10000 mm–2 s) under intact, global ischemic, and cold‐injury conditions. DWI was additionally applied to centrifuged human erythrocyte samples with a negligible extracellular space. Biexponential signal decay was found to occur in the cortex of the intact mouse brain. During global ischemia, in addition to a drop in the ADC in both components, a shift from the volume fraction of the rapidly diffusing component to the slowly diffusing one was observed. In cold injury, the biexponential signal decay was still present despite the electron‐microscopically validated disintegration of the membranes. The biexponential function was also applicable for fitting of the data obtained on erythrocyte samples. The results suggest that compartmentalization is not an essential feature of biexponential decay in diffusion experiments. Magn Reson Med 51:278–285, 2004.

Formation of "dark" (argyrophilic) neurons of various origin proceeds with a common mechanism of biophysical nature (a novel hypothesis).

SummaryBased on recent findings described in accompanying reports as well as on relevant observations in the literature we hypothesize that: (1) the fundamental elements in the mechanism of the formation of “dark” (argyrophilic) neurons are independent of the causative conditions including post-mortem or in vivo mechanical injuries and various in vivo pathometabolic processes such as blood recirculation following ischemia; (2) the causative conditions, each in its own mechanical or metabolic way, induce the same morphopathological damage at one point only within each affected neuron; (3) this damage spreads throughout the respective somato-dendritic or axonal domain and entails type III argyrophilia; (4) the intraneuronal spread of the morphopathological damage consumes mechanical energy stored by the neurofilaments in the form of a metastable inner structure, and (5) is propagated by a process working, in certain structural and energetical respects, on the domino principles; and (6) the primary neuronal damage caused in the above manner might be secondarily modified in different directions by different postcausation conditions.

BGP-15 — a novel poly(ADP-ribose) polymerase inhibitor — protects against nephrotoxicity of cisplatin without compromising its antitumor activity

Nephrotoxicity is one of the major dose limiting side effects of cisplatin chemotherapy. The antitumor and toxic effects are mediated in part by different mechanisms, thus, permitting a selective inhibition of certain side effects. The influence of O-(3-piperidino-2-hydroxy-1-propyl)nicotinic amidoxime (BGP-15) - a poly(ADP-ribose) polymerase (PARP) inhibitor - on the nephrotoxicity and antitumor efficacy of cisplatin has been evaluated in experimental models. BGP-15 either blocked or significantly reduced (60-90% in 100-200 mg/kg oral dose) cisplatin induced increase in serum urea and creatinine level in mice and rats and prevented the structural degeneration of the kidney, as well. The nephroprotective effect of BGP-15 treatment was revealed also in living mice by MRI analysis manifesting in the lack of oedema which otherwise developed as a result of cisplatin treatment. The protective effect was accompanied by inhibition of cisplatin-induced poly-ADP-ribosylation and by the restoration of the disturbed energy metabolism. The preservation of ATP level in the kidney was demonstrated in vivo by localized NMR spectroscopy. BGP-15 decreased cisplatin-induced ROS production in rat kidney mitochondria and improved the antioxidant status of the kidney in mice with cisplatin-induced nephropathy. In rat kidney, cisplatin caused a decrease in the level of Bcl-x, a mitochondrial protective protein, and this was normalized by BGP-15 treatment. On the other hand, BGP-15 did not inhibit the antitumor efficacy of cisplatin in cell culture and in transplantable solid tumors of mice. Treatment with BGP-15 increased the mean survival time of cisplatin-treated P-388 leukemia bearing mice from 13 to 19 days. PARP inhibitors have been demonstrated to diminish the consequences of free radical-induced damage, and this is related to the chemoprotective effect of BGP-15, a novel PARP inhibitor. Based on these results, we propose that BGP-15 represents a novel, non-thiol chemoprotective agent.

Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin, a Major Red Wine Polyphenol

Background Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways. Methods & Findings The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation. Conclusions These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease.