Mitochondrial protective effects of PARP-inhibition in hypertension-induced myocardial remodeling and in stressed cardiomyocytes.

AIMS: During oxidative stress mitochondria become the main source of endogenous reactive oxygen species (ROS) production. In the present study, we aimed to clarify the effects of pharmacological PARP-1 inhibition on mitochondrial function and quality control processes. MAIN METHODS: L-2286, a quinazoline-derivative PARP inhibitor, protects against cardiovascular remodeling and heart failure by favorable modulation of signaling routes. We examined the effects of PARP-1 inhibition on mitochondrial quality control processes and function in vivo and in vitro. Spontaneously hypertensive rats (SHRs) were treated with L-2286 or placebo. In the in vitro model, 150 µM H2O2 stress was applied on neonatal rat cardiomyocytes (NRCM). KEY FINDINGS: PARP-inhibition prevented the development of left ventricular hypertrophy in SHRs. The interfibrillar mitochondrial network were less fragmented, the average mitochondrial size was bigger and showed higher cristae density compared to untreated SHRs. Dynamin related protein 1 (Drp1) translocation and therefore the fission of mitochondria was inhibited by L-2286 treatment. Moreover, L-2286 treatment increased the amount of fusion proteins (Opa1, Mfn2), thus preserving structural stability. PARP-inhibition also preserved the mitochondrial genome integrity. In addition, the mitochondrial biogenesis was also enhanced due to L-2286 treatment, leading to an overall increase in the ATP production and improvement in survival of stressed cells. SIGNIFICANCE: Our results suggest that the modulation of mitochondrial dynamics and biogenesis can be a promising therapeutical target in hypertension-induced myocardial remodeling and heart failure.

High-throughput screening of saliva for early detection of oral cancer: a pilot study.

The success of tumour therapy depends considerably on early diagnosis. Therefore, we aimed to develop a widely available, cheap, non-invasive, high-throughput method suitable for screening high-risk populations, at least, for early signs of malignant transformation in the oral cavity. First, in order to identify suitable tumour marker candidates, we compared the protein patterns of five selected saliva samples obtained from healthy controls and tumour patients after electrophoretic separation, excised the bands that were consistently up-regulated in the tumour patients only, and performed matrix-assisted laser-desorption ionisation (MALDI)-time of flight (TOF) tandem mass spectrometry (MS/MS) analysis of the proteins in these bands after in-gel tryptic digestion. From the panel of proteins identified, we chose annexin 1 and peroxiredoxin 2 for further studies based on their presence in the saliva of all five oral cancer patients only. Then, we performed a homology search of protein databases using the primary sequence of each in silico tryptic fragment peptide of these two proteins as bait, and selected a unique peptide for each. Finally, we performed targeted MALDI-TOF MS peptide analysis in a blinded fashion on all samples obtained from 20 healthy controls and 22 tumour patients for the presence of these peptides. We found both peptides present in the saliva samples of all cancer patients only. Even though these tumour markers should be validated in a wider population, our results indicate that targeted MALDI-TOF MS analysis of unique peptides of putative saliva protein tumour biomarkers could be the method of choice for cost-efficient, high-throughput screening for the early detection of oral cancer.

A cytoplasmic gel network capable of mediating the conversion of chemical energy to mechanical work in diverse cell processes: a speculation.

Enigmatic morphological features of the formation and fate of "dark" (hyper-basophilic, hyper-argyrophilic and hyper-electrondense) neurons suggest that the mechanical work causing their dramatic shrinkage (whole-cell ultrastructural compaction) is done by a previously "unknown" ultrastructural component residing in the spaces between their "known" (i.e. visible in the conventional transmission electron microscopy) ultrastructural constituents. Embedment-free section electron microscopy revealed in these spaces the existence of a continuous network of gel microdomains, which is embedded in a continuous network of fluid-filled lacunae. We gathered experimental facts suggesting that this gel network is capable of a volume-reducing phase-transition (an established physico-chemical phenomenon), which could be the motor of the whole-cell ultrastructural compaction. The present paper revisits our relevant observations and speculates how such a continuous whole-cell gel network can do both whole-cell and compartmentalized mechanical work.

Pituitary adenylate cyclase activating polypeptide in the retina: focus on the retinoprotective effects.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects against different neuronal injuries, such as traumatic brain and spinal cord injury, models of neurodegenerative diseases, and cerebral ischemia. PACAP and its receptors are present in the retina. In this study, we summarize the current knowledge on retinal PACAP with focus on the retinoprotective effects. Results of histological, immunohistochemical, and molecular biological analysis are reviewed. In vitro, PACAP shows protection against glutamate, thapsigargin, anisomycin, and anoxia. In vivo, the protective effects of intravitreal PACAP treatment have been shown in the following models of retinal degeneration in rats: excitotoxic injury induced by glutamate and kainate, ischemic injury, degeneration caused by UV-A light, optic nerve transection, and streptozotocin-induced diabetic retinopathy. Studying the molecular mechanism has revealed that PACAP acts by activating antiapoptotic and inhibiting proapoptotic signaling pathways in the retina in vivo. These studies strongly suggest that PACAP is an excellent candidate retinoprotective agent that could be a potential therapeutic substance in various retinal diseases.

TIP47 protects mitochondrial membrane integrity and inhibits oxidative-stress-induced cell death.

We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t-TIP47) protected NIH3T3 cells from hydrogen-peroxide-induced cell death, prevented the hydrogen-peroxide-induced mitochondrial depolarization determined by 5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative-stress-induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t-TIP47 increased the mitochondrial membrane potential (Deltapsi), and partially prevented Ca2+ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.

Effects of PACAP and preconditioning against ischemia/reperfusion-induced cardiomyocyte apoptosis in vitro.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) and its receptors are widely expressed in the nervous system and various other tissues. PACAP exerts strong anti-apoptotic effects in neuronal cell lines and, according to recent data, also in non-neuronal cells. The peptide is present in the cardiovascular system and has various distinct effects. We have demonstrated earlier that PACAP has protective effects against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Preconditioning with brief intermittent periods of ischemia is known to provide protection against ischemic injury. The aim of the present study was to investigate whether PACAP could enhance the protective effect of preconditioning against in vitro ischemic injury. Cultured cardiomyocytes were exposed to brief preconditioning ischemia followed by 2 h ischemia and 4 h reperfusion. Both PACAP treatment and preconditioning alone significantly increased cell viability and decreased the ratio of cell death. Pretreatment with PACAP was found to further reduce the level of cleaved caspase-8 but it did not lead to additional survival rate when compared to cells treated with PACAP or preconditioning alone. These results show that although both PACAP and preconditioning have a protective effect against ischemia/reperfusion-induced cardiomyocyte apoptosis, their effects are not additive.

Physicochemical mechanisms of histological silver staining and their utilization for rendering individual silver methods selective and reliable.

Staining morphological or chemical constituents of biological tissues, cells and microorganisms with silver proceeds via different reaction routes. In this paper, I put their physicochemical mechanisms into a coherent system and discuss how these can be controlled and separated from each other, thereby permitting selective, sensitive and reliable demonstration of individual tissue constituents.

PKA-Bad-14-3-3 and Akt-Bad-14-3-3 signaling pathways are involved in the protective effects of PACAP against ischemia/reperfusion-induced cardiomyocyte apoptosis.

The neuropeptide PACAP (pituitary adenylate cyclase activating polypeptide) and its receptors are widely expressed in the nervous system and various other tissues. PACAP has well-known anti-apoptotic effects in neuronal cell lines. Recent data suggest that PACAP exerts anti-apoptotic effects also in non-neuronal cells. The peptide is present in the cardiovascular system, and has various distinct effects. The aim of the present study was to investigate whether PACAP is protective against in vitro ischemia/reperfusion-induced apoptosis in cardiomyocytes. Cultured cardiomyocytes were exposed to 60 min ischemia followed by 120 min reperfusion. The addition of PACAP1-38 significantly increased cell viability and decreased the ratio of apoptotic cells as measured by MTT test and flow cytometry. PACAP induced the phosphorylation of Akt and protein kinase A. In the present study we also examined the possible involvement of Akt- and protein kinase A-induced phosphorylation and thus inactivation of Bad, a pro-apoptotic member of the Bcl-2 family. It was found that ischemia significantly decreased the levels of phosphorylated Bad, which was counteracted by PACAP. Furthermore, PACAP increased the levels of Bcl-xL and 14-3-3 protein, both of which promote cell survival, and decreased the apoptosis executor caspase-3 cleavage. All effects of PACAP1-38 were inhibited by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP has protective effects against ischemia/reperfusion-induced cardiomyocyte apoptosis and provides new insights into the signaling mechanisms involved in the PACAP-mediated anti-apoptotic effects.

Protective effects of pituitary adenylate cyclase activating polypeptide in endothelial cells against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a widely distributed neuropeptide that has various different functions in the nervous system and in non-neural tissues. Little is known about the effects of PACAP in endothelial cells. The aim of the present study was to investigate the effects of PACAP on endothelial cell survival and apoptotic signaling pathways under oxidative stress. Mouse hemangioendothelioma (EOMA) cells were exposed to 0.5mM H(2)O(2) which resulted in a marked reduction of cell viability and a parallel increase of apoptotic cells assessed by MTT test and flow cytometry. Co-incubation with 20nM PACAP1-38 increased cell viability and reduced the percentage of apoptotic cells. Flow cytometry analysis showed that oxidative stress reduced the phosphorylation of the anti-apoptotic ERK and increased the phosphorylation of the pro-apoptotic JNK and p38 MAP kinases. PACAP1-38 treatment ameliorated these changes: levels of phospho-ERK were elevated and those of phospho-JNK and p38 were decreased. All these effects were abolished by simultaneous treatment with the PACAP antagonist PACAP6-38. In summary, our results show that PACAP effectively protects endothelial cells against the apoptosis-inducing effects of oxidative stress.

Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis.

Pituitary adenylate cyclase activating polypeptide (PACAP) has well-known neuroprotective effects, and one of the main factors leading to neuroprotection seems to be its anti-apoptotic effects. The peptide and its receptors are present also in the heart, but whether PACAP can be protective in cardiomyocytes, is not known. Therefore, the aim of the present study was to investigate the effects of PACAP on oxidative stress-induced apoptosis in cardiomyocytes. Our results show that PACAP increased cell viability by attenuating H2O2-induced apoptosis in a cardiac myocyte culture. PACAP also decreased caspase-3 activity and increased the expression of the anti-apoptotic markers Bcl-2 and phospho-Bad. These effects of PACAP were counteracted by the PACAP antagonist PACAP6-38. In summary, our results show that PACAP is able to attenuate oxidative stress-induced cardiomyocyte apoptosis.

Recovery versus death of "dark" (compacted) neurons in non-impaired parenchymal environment: light and electron microscopic observations.

The formation of massively shrunken, hyperbasophilic, hyperargyrophilic and hyper-electron-dense but not apoptotic ("dark") neurons was initiated in rat brains by means of an electric-shock and two mechanical-injury paradigms that do not cause considerable parenchymal damage in the areas investigated. The rats were killed by perfusion fixation either immediately after these instantaneous initiating insults or after a survival period ranging from 40 min to 6 days. The formation of "dark" neurons was complete in less than a few minutes. In the somatodendritic domain of each "dark" neuron, all ultrastructural elements were remarkably preserved during the acute stage, apart from a dramatic reduction of the distances between them. This ultrastructural compaction was accompanied by a marked shift of cell fluid through seemingly intact plasma membrane, mainly into surrounding astrocytic elements. The majority of the "dark" neurons regained their normal morphology and staining properties (recovery) in 4 h. Thereafter, only solitary mitochondrion-derived membranous whorls in the cytoplasm reminded of a previous morphological disturbance. The dead "dark" neurons fell apart into membrane-bound fragments that retained their sharp outlines and compacted interior even after being engulfed by astrocytes or microglial cells. The latter sequence of morphological changes can not be harmonized with the prevailing assumption, according to which "dark" neurons die through the necrotic pathway. The fate of the "dark" neurons appeared to depend on the presence or absence of serious post-insult pathophysiological circumstances in their surroundings.

Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance.

It was earlier established that one of the primary morphopathological consequences of experimental traumatic brain injury is a dramatic reduction in the distances between the neurofilaments (cytoskeletal compaction) inside a number of axon segments that appear to be randomly distributed among normal axons in an otherwise undamaged parenchymal environment. The present results demonstrate that the cytoskeletal compaction instantly induces argyrophilia, thereby rendering possible selective visualisation of the affected axon segments for light microscopy through use of a special silver staining method. On combination of this method with electron microscopy, it was revealed that the cytoskeletal compaction is completed in much shorter times and extends to much longer axon segments than previously assumed.

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.

Granule cells are the main source of excitatory input to a subpopulation of GABAergic hippocampal neurons as revealed by electron microscopic double staining for zinc histochemistry and parvalbumin immunocytochemistry.

Immunocytochemistry was combined with a recent modification of Timm's method to evaluate semiquantitatively the mossy fiber innervation of dendrites and somata of parvalbumin-containing neurons of the hilus of the dentate gyrus and the CA3 area of Ammon's horn. Using this electron microscopic double staining technique, it was found that (1) the overwhelming majority (95%) of terminals forming asymmetric synapses with parvalbumin-positive dendrites in the dentate hilus, and the strata pyramidale and lucidum of the CA3 area of Ammon's horn, originated from granule cells; (2) two-thirds of the asymmetric axosomatic terminals of parvalbumin-positive neurons contained zinc; and (3) no zinc-containing axon terminals formed synapses with somata or main dendritic shafts of the granule cells.

The use of a sodium tungstate developer markedly improves the electron microscopic localization of zinc by the Timm method.

The Timm's sulfide-silver method is frequently used for the demonstration of the mossy fiber bundle or sprouted mossy fibers in the normal or epileptic hippocampal dentate gyrus. Under the light microscope the results are excellent, but the ultrastructure is considerably impaired and the silver grains produced are too large as compared to the sizes of intra-synaptic structures. The present study was meant to test a series of physical developers containing, instead of gum arabic, sodium tungstate as protective colloid. One of them left the ultrastructure fairly intact and produced small, round silver grains, making it possible to precisely locate zinc in mossy terminals. With this method, it could be demonstrated that zinc is contained inside synaptic vesicles in the resting axon terminals of granule cells. As a consequence of prolonged sodium sulfide perfusion, zinc is released from synaptic vesicles and enters the synaptic cleft.

Enhanced ADP-ribosylation and its diminution by lipoamide after ischemia-reperfusion in perfused rat heart.

Poly-ADP-ribose polymerase (PARP) is considered to play an important role in oxidative cell damage. We assumed that ischemia-reperfusion resulting from the increasing reactive oxygen species (ROS) can lead to the activation of endogenous mono- and poly-ADP-ribosylation reactions and that the reduction of ROS level by lipoamide, a less known antioxidant, can reverse these unfavorable processes. Experiments were performed on isolated Langendorff hearts subjected to 60-min ischemia followed by reperfusion. ROS, malondialdehyde, deoxyribonucleic acid (DNA) breaks, and NAD+ content were assayed in the hearts, and the ADP-ribosylation of cytoplasmic and nuclear proteins were determined by Western blot assay. Ischemia-reperfusion caused a moderate (30.2 +/- 8%) increase in ROS production determined by the dihydrorhodamine 123 method and significantly increased the malondialdehyde production (from < 1 to 23 +/- 2.7 nmol/ml), DNA damage (undamaged DNA decreased from 71 +/- 7% to 23.1 +/- 5%), and NAD+ catabolism. In addition, ischemia-reperfusion activated the mono-ADP-ribosylation of GRP78 and the self-ADP-ribosylation of the nuclear PARP. The perfusion of hearts with lipoamide significantly decreased the ischemia-reperfusion-induced cell membrane damage determined by enzyme release (LDH, CK, and GOT), decreased the ROS production, reduced the malondialdehyde production to 5.5 +/- 2.4 nmol/ml, abolished DNA damage, and reduced NAD+ catabolism. The ischemia-reperfusion-induced activation of poly- and mono-ADP-ribosylation reactions were also reverted by lipoamide. In isolated rat heart mitochondria, dihydrolipoamide was found to be a better antioxidant than dihydrolipoic acid. Ischemia-reperfusion by ROS overproduction and increasing DNA breaks activates PARP leading to accelerated NAD+ catabolism, impaired energy metabolism, and cell damage. Lipoamide by reducing ROS levels halts PARP activation and membrane damage and improves the recovery of postischemic myocardium.

Initial clinical experience with a combined pulsed holmium-neodymium-YAG laser in minimally invasive neurosurgery.

Various biophysical features of the laser beam have already been utilized in clinical neurosurgery. However, the application of this therapeutic modality has by no means been overexploited. The history of laser application in neurosurgery has shown that there is no universal laser system capable of performing all surgical tasks in a suitable manner. The best results in traditional neurosurgery were achieved with instruments combining various wavelengths, such as the CO2 and neodymium-YAG lasers. A pulsed holmium-YAG and neodymium-YAG (Ho:YAG and Nd:YAG) combined laser have been recently developed to meet the special requirements of minimally invasive neurosurgery. The system consists of a compact double-crystal single-head solid-state laser system generating 2 different wavelengths (Ho:YAG 2.08 microns and Nd:YAG 1.05 microns), selected for their capabilities of efficient coagulation and ablation. The two wavelengths are coupled into a common flexible optical fiber, which allows endoscopic application. The wavelengths can act simultaneously or separately without any interchange of the instruments. The system was employed first for experimental and subsequently for clinical purposes, primarily for endoscopic operations. In this work the initial clinical experience is reported. The excellent haemostatic properties of the Nd:YAG laser and the ablative properties of the Ho:YAG laser were confirmed. It was concluded that simultaneous application of the two laser modalities within one flexible fiber offers new perspectives in tissue handling in endoscopic neurosurgery and as in open microsurgery.

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of A beta1-42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited A beta42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.

Identifying monoaminergic, GABAergic, and cholinergic characteristics in immortalized neuronal cell lines.

We measured the concentration of neurotransmitters in immortalized neural cell lines of hippocampal, septal, brainstem and cerebellar origin. While in most of the cell lines, concentrations of monoamines, gamma-aminobutyric acid (GABA) and acetylcholine were low, in some they were markedly higher. This made it quite easy to identify possible monoaminergic, GABAergic or cholinergic cell lines. However all the cell lines contained glutamate and aspartate and there were no outstanding differences in levels of these amino acids differences between the cell lines. Deprivation of serum, which made the cells acquire a more differentiated morphology, caused an increase in the intracellular concentrations of some compounds and a switch from multiple to a single transmitter in the case of some cell lines. It suggested that measurement of transmitter concentrations combined with serum deprivation studies, may provide an indication of the neurochemical characteristics of immortalised neuronal cell lines.

A common morphological response of astrocytes to various injuries: "dark" astrocytes. A light and electron microscopic analysis.

Our previous studies showed that neurons became argyrophilic following different kinds of brain injuries. In the present study we demonstrated that astrocytes could also become argyrophilic following compressive or concussive head injuries and following intraperitoneal administration of pentylenetetrazole or kainic acid. Furthermore the soma, nucleus and processes of these argyrophilic astrocytes were shown in other preparations to be hyper-basophilic with the light microscope, and hyper-electron dense and shrunken at the ultrastructural level. When the head injuries were inflicted either at the 15th min. of perfusion-fixation or at the 30th min. of transcardial perfusion with chilled physiological saline, several astrocytes also became argyrophilic, hyper-basophilic, shrunken and hyper-electron dense. These data indicate that (i) the intracellular pathological event in these astrocytes is similar to that of "dark" neurons or "dark" cells of non-neural tissues, (ii) the formation of "dark" astrocytes can be independent of the actual state of metabolism, and (iii) the "dark" morphological state of astrocytes might have a role in neuropathological processes.