Consequences of metaphase II oocyte cryopreservation on mRNA content.

INTRODUCTION: We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). MATERIAL AND METHODS: Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. RESULTS: There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. CONCLUSIONS: Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte.

Cyanidin and cyanidin 3-O-beta-D -glucoside as DNA cleavage protectors and antioxidants.

Anthocyanins, colored flavonoids, are water-soluble pigments present in the plant kingdom; in fact they are secondary plant metabolites responsible for the blue, purple, and red color of many plant tissues. Present in beans, fruits, vegetables and red wines, considerable amounts of anthocyanins are ingested as constituents of the human diet (180-215 mg daily). There is now increasing interest in the in vivo protective function of natural antioxidants contained in dietary plants against oxidative damage caused by free radical species. Recently, the antioxidant activity of phenolic phytochemicals, has been investigated. Since the antioxidant mechanism of anthocyanin pigments is still controversial, in the present study we evaluated the effects of cyanidin and cyanidin 3-O-beta-D-glucoside on DNA cleavage, on their free radical scavenging capacity and on xanthine oxidase activity. Cyanidin and cyanidin 3-O-beta-D-glucoside showed a protective effect on DNA cleavage, a dose-dependent free radical scavenging activity and significant inhibition of XO activity. These effects suggest that anthocyanins exhibit interesting antioxidant properties, and could therefore represent a promising class of compounds useful in the treatment of pathologies where free radical production plays a key role.

DNA damage in astrocytes exposed to fumonisin B1.

Fumonisins are a group of toxic metabolites mainly produced by Fusarium moniliforme and Fusarium proliferatum, fungi that commonly occur on corn throughout the world. Fumonisin B1 (FB1), structurally resembling sphingoid bases, is an inhibitor of ceramide synthase, a key enzyme involved in de novo sphingolipid biosynthesis and in the reacylation of free sphingoid bases derived from sphingolipid turnover. This inhibitory effect leads to accumulation of free sphinganine (SA) and sphingosine (SO), inducing cell death. However, little is known on the down stream effectors activated by these sphingolipids in the cell death signaling pathway. We exposed rat astrocytes to FB1 with the aim of evaluating the involvement of oxygen free radicals and of some other biochemical pathways such as caspase-3 activity and DNA damage. Our results indicate that FB1 treatment (48, 72 h and 6 days in vitro, DIV, and 10, 50, 100 microM) does not affect cell viability. Conversely, after 72 h of treatment, FB1 (50 and 100 microM) induced DNA damage and an enhancement of caspase-3 activity compared to controls. In addition, FB1 increased the expression of HSP70 at 10 and 50 microM at 48, 72 h, and 6 DIV of treatment. We conclude that DNA damage of apoptotic type in rat astrocytes is caused by FB1 and that the genotoxic potential of FB1 has probably been underestimated and should be reconsidered.

Ethanol-induced oxidative stress in rat astrocytes: role of HSP70.

Ethanol intake is associated with increase in lipid peroxidation and formation of reactive oxygen species in different cerebral areas, in neurons as well as in astrocytes. The latter's integrity is essential for the normal growth of neurons. In previous studies we observed, in different cerebral areas of both acutely and chronically ethanol-treated rats, correlation between ethanol-induced oxidative stress and the increased expression of HSP70 (70 kDa heat shock proteins), chaperonins having a protective and stabilizing effect on stress-induced cell injury. In this study we examined, in vitro, the role of HSP70 on chronically ethanol-treated rat astrocytes by transfection with an anti-HSP70 antisense oligonucleotide. The results show that treatment with ethanol, from 50 to 100 mmol/L, induces a dose-dependent increase in the production of reactive oxygen species and of HSP70 levels, together with an impairment of the respiratory chain activity and a decrease in cell viability. In addition, our data indicate a drastic reduction of cellular metabolism in HSP70-deprived astrocytes, particularly when these cells were also ethanol-treated. In fact, transfection with HSP70 antisense induced moderate oxidative damage in control astrocytes and, consequently, a drastic decrease in the viability of ethanol-treated cells, with the mitochondrial functionality being particularly affected. Our results confirm that heat shock proteins confer a survival advantage to the astrocytes, preventing oxidative damage and nuclear DNA damage as well, and suggest the development of new drugs exerting a cytoprotective role either in physiological, or pathological conditions.

4',6-diamidino-2-phenylindole (DAPI) interacts with rare structures of GC polymers.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to double-stranded GC polymers either in the alternating or in homopolymer sequence was investigated using fluorescence techniques. We employed fluctuation correlation spectroscopy, which measures the diffusion coefficient of fluorescent particles, to demonstrate that the fluorescence was originating from relatively slowly diffusing entities. These entities display a very large heterogeneity of diffusing coefficients, indicating that molecular aggregation is extensive in our samples. We used frequency domain fluorometry to characterize the fluorescence lifetime of the species, while varying the GC polymer-dye coverage systematically. At very low coverage we observed a relatively bright fluorescent component with a lifetime value of approximately 4 ns. The stoichiometry of binding of this bright species was such that it can only arise from rare molecular structures, either unusual loops or large molecular aggregates. The amount and characteristics of this bright fluorescent component were different between the homo and the alternating polymer, indicating that the difference in sequence of the two polymers is responsible for the different aggregates which are then detected in the fluorescence experiment. At large GC polymer coverage we observed a relatively wide distribution of fluorescent species with short lifetime values, in the range between 0.12 and 0.2 ns. Given the stoichiometry of binding of this fluorescent component, we concluded that it could arise either from intercalative and/or non-specific binding to the DNA double-stranded molecules. We comment on the origin of the rare but brightly fluorescent binding sites and discuss the potential to detect such unusual DNA structures.

Fibroblast growth factor-2 and transforming growth factor-beta1 immunostaining in rat brain after cerebral postischemic reperfusion.

Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor-2 (FGF-2) plays an important role in the "self-repair" responses that follow injuries such as trauma and brain ischemia and that FGF-2 contributes to the repair of damaged tissue. Transforming growth factor-beta (TGF-beta) is a potent growth-regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF-2 and TGF-beta1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF-beta1 and FGF-2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron-glia interaction but also to neurochemical conditions involving inhibitory interneurons.

Bioflavonoids as antiradicals, antioxidants and DNA cleavage protectors.

Flavonoids have recently aroused considerable interest because of their broad pharmacological activity. In fact, flavonoids have been reported to have antiviral, antiallergic, antiplatelet, anti-inflammatory and antitumoral activities. The pharmacological properties of bioflavonoids have been ascribed both to the concomitant inhibition of enzymes involved in the production of free radicals and to their free-radical scavenging and iron chelating capacity. However the antioxidant capacity of bioflavonoids due to free-radical scavenging and/or to iron chelating is still controversial. In this study, we have investigated the free-radical scavenging capacity of bioflavonoids (rutin, catechin, and naringin). In addition, the effects of these polyphenols on xanthine oxidase activity, spontaneous lipid peroxidation, and DNA cleavage were investigated. The bioflavonoids under examination showed a dose-dependent free-radical scavenging effect, a significant inhibition of xanthine oxidase activity, and an antilipoperoxidative capacity. In addition, they showed a protective effect on DNA cleavage.

L -propionyl-carnitine as superoxide scavenger, antioxidant, and DNA cleavage protector.

L-Propionylcarnitine, a propionyl ester of L-carnitine, increases the intracellular pool of L-carnitine. It exhibits a high affinity for the enzyme carnitine acetyltransferase (CAT) and, thus, is readily converted into propionyl-coenzyme A and free carnitine. It has been reported that L-propionylcarnitine possesses a protective action against heart ischemia-reperfusion injury; however, the antioxidant mechanism is not yet clear. L-Propionylcarnitine might reduce the hydroxyl radical production in the Fenton system, by chelating the iron required for the generation of hydroxyl radicals. To obtain a better insight into the antiradical mechanism of L-propionylcarnitine, the present research analyzed the superoxide scavenging capacity of L-propionylcarnitine and its effect on linoleic acid peroxidation. In addition, the effect of L-propionylcarnitine against DNA cleavage was estimated using pBR322 plasmid. We found that L-propionylcarnitine showed a dose-dependent free-radical scavenging activity. In fact, it was able to scavenge superoxide anion, to inhibit the lipoperoxidation of linoleic acid, and to protect pBR322 DNA from cleavage induced by H2O2 UV-photolysis.

Stress proteins and SH-groups in oxidant-induced cellular injury after chronic ethanol administration in rat.

It is generally agreed that lipid peroxides play an important role in the pathogenesis of ethanol-induced cellular injury and that free sulfhydryl groups are vital in cellular defense against endogenous or exogenous oxidants. It has been observed that oxidative stress induces the synthesis of the 70-kDa family of heat-shock proteins (HSPs). Induction of HSPs represents an essential and highly conserved cellular response to a variety of stressful stimuli. In the present study we measured in various brain areas and in liver the intracellular levels of HSP70 proteins, sulfhydryl groups and the antioxidant enzyme status after chronic administration of mild intoxicating doses of ethanol to rats. Expression of HSP70 in response to alcohol administration was particularly high in the hippocampus and striatum. In these brain areas, the increase in HSP70 protein levels occurred in absence of significant changes of antioxidant enzyme activities and was correlated with a marked depletion of intracellular bound thiols and with a decreased susceptibility to lipid peroxidation. Lower levels of HSP70 induction were found in cortex and cerebellum and were associated to decreases in SOD and CAT enzyme activities, with a lower depletion of protein bound thiols and with an increased susceptibility to lipid peroxidation. This study agrees with our previous results performed on acute alcohol intoxication and supports the hypothesis that HSP70 induction protects the different brain areas against oxidative stress.

MAP2, synaptophysin immunostaining in rat brain and behavioral modifications after cerebral postischemic reperfusion.

Plasticity in the central nervous system after cerebral ischemia is a controversial issue; focal cerebral ischemia produces an area of infarction that is surrounded by neurons that may respond to nearby damage by creating new synapses. In the present study the expression of the postsynaptic microtubule-associated protein 2 (MAP2) and the presynaptic marker protein, synaptophysin, was investigated by immunocytochemical techniques in the CA1 sector of hippocampus and in cerebellum of rats made ischemic by bilateral clamping of common carotid arteries and reperfused for 7 and 30 days. In addition, ischemia-induced behavioral alterations were also evaluated after 7 and 30 days of reperfusion. The present study demonstrates a decreased postsynaptic MAP2 immunoreactivity, representative of neuronal loss, particularly in CA1 sector of hippocampus and in cerebellum of ischemic rats reperfused for 7 days. After 30 days of reperfusion, MAP2 immunostaining was similar to control. In the same brain sections an increased presynaptic synaptophysin immunoreactivity has been observed only after 30 days of reperfusion. These data suggest compensatory regenerative changes associated with synaptic remodelling and are supported by behavioral recovery observed under the same experimental conditions.

Nuclear DNA strand breaks during ethanol-induced oxidative stress in rat brain.

Free radical-mediated oxidative damage has been implicated in the pathophysiological mechanisms of apoptosis. In this study we report that statistically significant strand breaks were induced primarily in the hippocampus and cerebellum during chronic, and not acute, ethanol treatment. Damage to DNA observed in hippocampus and cerebellum was also correlated with significant modification in the activities of mitochondrial respiratory complexes I and IV and with a significant increase in lipid peroxidation products. This finding lends support to the fact that hippocampus and cerebellum are brain areas particularly vulnerable to redox changes induced by alcohol intoxication, suggesting lower threshold levels of ethanol tolerance.

Fluorescence anisotropy of DNA/DAPI complex: torsional dynamics and geometry of the complex.

Fluorescence depolarization of synthetic polydeoxynucleotide/4'-6-diamidino-2-phenylindole dihydrochloride complexes has been investigated as a function of dye/polymer coverage. At low coverage, fluorescence depolarization is due to local torsional motions of the DNA segment where the dye resides. At relatively high coverage, fluorescence depolarization is dominated by energy transfer to other dye molecules along the DNA. The extent of the observed depolarization due to torsional motion depends on the angle the dye molecule forms with the DNA helical axis. A large torsional motion and a small angle produce the same depolarization as a small torsional motion and a large projection angle. Furthermore, the extent of transfer critically depends on the relative orientation of dye molecules along the DNA. The effect of multiple transfer is examined using a Monte Carlo approach. The measurement of depolarization with transfer, at high coverage, allows determination of the dye orientation about the DNA helical axis. The value of the torsional spring constant is then determined, at very low coverage, for few selected polydeoxynucleotides.

Torsional dynamics and orientation of DNA--DAPI complexes.

The flexibility of calf thymus DNA and several polynucleotides was measured using the anisotropy decay of DAPI bound to DNA, a minor groove probe. DNA torsional dynamics were analyzed using the Schurr model [Allison, S. A., & Schurr, J. M. (1979) Chem. Phys. 41, 35-44] in the infinite polymer length approximation. Time-resolved fluorescence depolarization was measured using a frequency-double mode-locked dye laser and frequency-domain acquisition methods. At very high P/D ratios, the anisotropy decay is dominated by DNA torsional dynamics. The recovered values of the torsional elastic constant were in good agreement with literature values obtained using other DNA probes. The exact knowledge of the angle between the probe emission dipole transition moment and the helix axis is critical for the determination of the polymer elastic constant. At low P/D ratios, energy transfer between dye molecules strongly contributes to the anisotropy decay. We have developed a statistical model that describes the anisotropy decay, when the correct geometrical factors are included. At low P/D ratios the anisotropy decay is dominated by fluorescence homotransfer. In this regime, it is possible to determine the orientation of the dye molecule with respect to the polymer with accuracy. The values obtained for the distance and orientation of the DAPI molecules in solution using the fluorescence measurements are in excellent agreement with those from the crystal structure of the oligonucleotides-DAPI complex by Dickerson's group [Larsen T.A., Goodsell, D. S., Cascio, D., Grzeskowiak, K., & Dickerson, R. E. (1989) J. Biomol. Struct. Dyn. 7, 477-491].

Stress proteins and SH-groups in oxidant-induced cell damage after acute ethanol administration in rat.

It is generally accepted that lipid peroxides play an important role in the pathogenesis of ethanol-induced cellular injury and that free sulfhydryl groups are vital in cellular defense against endogenous or exogenous oxidants. It has been observed that oxidative stress induces the synthesis of the 70-kDa family of heat-shock proteins (HSPs). Furthermore, induction of HSPs represents an essential and highly conserved cellular response to a variety of stressful stimuli. In the present study, we measured the intracellular levels of HSP 70 proteins after administration of mild intoxicating and grossly intoxicating doses of ethanol to rats. Our results demonstrate that elevated doses of ethanol induce HSP in various brain areas, namely, cerebellum, hippocampus, and to a lesser extent, striatum or liver. Induction of HSP 70 protein was correlated with a marked depletion of intracellular bound thiols and a decrease in lipid peroxidation measured as MDA formation. These studies support the hypothesis that a redox mechanism may be involved in the heat-shock signal pathway.

Heat shock proteins following rat cerebral ischemic/reperfusion episode: effect of ketamine.

This paper reports the influence of ketamine on HSP70 expression, during an ischemic/reperfusion episode, in rat cerebral cortex and hippocampus. The results indicate that ketamine, injected 1h before the surgical treatment, increases HSP70 cellular concentration in both ischemic and sham-operated animals. The HSP70 levels, after the transient ischemic episode, are higher in ketamine treated than in urethane-treated animals respect to the control levels. After reperfusion an increase of HSP70 levels is observed; this induction is maintained for at least 22h, irrespective of the anaesthetic drug treatment. Comparing the cerebral areas examined, the hippocampus exhibits higher protein levels than those of the cerebral cortex.

A molecular approach to 4',6-diamidine-2-phenylindole (DAPI) photophysical behaviour at different pH values.

The photophysical mechanisms which determine the spectral properties and decay rates of 4',6-diamidine-2-phenylindole (DAPI) in solution and in association with nucleic acids have not yet been fully elucidated. We have performed steady-state and time-resolved fluorescence experiments on DAPI in a wide pH range to investigate the hypothesis that different ground-state conformations are responsible for the photophysical properties of the probe. Several excited-state mechanisms are investigated and it is concluded that among the proposed models, the hypothesis of ground-state heterogeneity with rapid interconversion among conformations is the only one consistent with the experiments in the entire pH range investigated.

Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides.

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.

The fluorescence properties of a DNA probe. 4'-6-Diamidino-2-phenylindole (DAPI).

Steady-state and dynamic fluorescence measurements have been performed on DAPI in solution and in complexes formed with a number of synthetic and natural polydeoxynucleotides. The decay of DAPI in buffer at pH 7 was decomposed using two exponentials having lifetime values of approximately 2.8 ns and 0.2 ns. The double exponential character of the decay was maintained over a large pH range from 3 to 9. At pH 1 the short component dominated, whereas at pH 12, only the long component was detectable. Two distinct spectra were associated with the two lifetime components; the short component was shifted to the red. The short lifetime component occurs in the presence of water. In water the excitation spectra depended on the emission wavelength and there was no viscosity dependence of the two forms. To explain these results we propose that there is a ground state conformer in which preferential solvation of the indole ring allows proton transfer in the excited state. DAPI complexed with polydeoxynucleotides retained most of the features of the decay of DAPI in solution. However, the complexes with fully AT-containing polymers stabilized the longer lifetime form of DAPI because the stronger binding enhanced solvent shielding. A gradual increase of the short lifetime component, which monitors dye solvent exposure, was obtained as the AT content was decreased. For polyd(GC) the decay was similar to that of free DAPI.

Fluorescence lifetime distributions of DNA-4',6-diamidino-2-phenylindole complex.

Time-resolved fluorescence of 4',6-diamidino-2-phenylindole (DAPI) complexes show that for a homogeneous polymer (polyd(AT) or polyd(A).polyd(T)) at high P/D (phosphate/dye) ratio, a single exponential component adequately describes the fluorescence decay. For the AT polymers at low P/D ratio or for native DNA, the decay cannot be described by a single-exponential term. A continuous distribution of lifetime values of Gaussian shape gives a good fit to the decay data. We propose that the lifetime distribution method for the analysis of the fluorescence decay of DNA-DAPI complexes provides a useful method of characterizing the microheterogeneity of site binding.