Age-dependent changes in the expression of superoxide dismutases and catalase are associated with ultrastructural modifications in human granulosa cells.

Limited knowledge exists about changes in follicle quality associated with age. The aim of this work was to investigate whether ageing may cause oxidative stress-mediated alterations in human granulosa cells (GCs) from periovulatory follicles. GCs employed in this study were obtained from follicular aspirates of 20 younger women (range 27-32 years) and 20 older women (range 38-41 years) undergoing an IVF treatment. Results obtained from comparative RT-PCR analysis revealed that the mean relative levels of mRNAs coding for superoxide dismutases, Cu, ZnSOD (SOD1), MnSOD (SOD2) and catalase were significantly decreased in women > or =38 years (P < 0.05, Student's t-test). These changes were associated with a reduced expression of SOD1, SOD2 and catalase at the protein level. When examined at an ultrastructural level, most of the GCs from this group showed defective mitochondria and fewer lipid droplets than those observed in the younger group. These results indicate that GCs from older patients suffer from age-dependent oxidative stress injury and are taken as an evidence for reduced defence against reactive oxygen species (ROS) in GCs during reproductive ageing.

Peroxisome proliferator-activated receptors (PPARs) and related transcription factors in differentiating astrocyte cultures.

Peroxisome proliferator-activated receptors (PPARs), retinoid X receptors (RXRs), CCAAT/enhancer binding proteins (C/EBPs) and beta-catenin are transcription factors involved in cell differentiation. The aim of this work was to investigate the occurrence and variations of these proteins during astrocyte differentiation. Primary cultures of mouse cortical astrocytes were characterized using nestin, A2B5 and glial fibrillary acidic protein (GFAP) as differentiation markers, during a period of 21 days in vitro (DIV). Glycogen and triglyceride accumulation were also studied. At 3 DIV the cultures were mainly constituted by neural progenitor cells, as assessed by their immunofluorescent pattern. At this time PPARs and beta-catenin were localized to the cytoplasm. Interestingly, some cells contained Oil Red O-positive lipid droplets. Between 7 and 21 DIV, nestin decreased, while GFAP increased, indicating ongoing astroglial differentiation. beta-catenin, predominantly nuclear at 7 DIV, later localized to membranes. Redistribution of all three PPAR isotypes from the cytoplasm to the nucleus was observed starting from 7 DIV. Between 7 and 14 DIV, C/EBPalpha, PPARalpha, RXRalpha and glycogen content increased. Between 14 and 21 DIV, PPARbeta/delta decreased, while PPARgamma, C/EBPbeta and delta and lipid droplet-containing cells increased. At 21 DIV both A2B5-/GFAP+ and A2B5+/GFAP+ cells were predominantly observed, indicating differentiation toward type-1 and type-2 astrocytes, although the presence of GFAP- cells demonstrates the persistence of neural precursors in the culture even at this time point. In conclusion, our results, reporting modifications of PPARs, RXRs, C/EBPs and beta-catenin during culture time, strongly suggest the involvement of these transcription factors in astrocyte differentiation. Specifically, beta-catenin translocation from the nucleus to plasma membrane, together with PPARbeta/delta decrease and C/EBPalpha increase, could be related to decreased proliferation at confluence, while PPARalpha and gamma and all C/EBPs could participate in differentiation processes, such as glycogenesis and lipidogenesis.

Comparative analysis of isolated cellular organelles by means of soft X-ray contact microscopy with laser-plasma source and transmission electron microscopy.

Soft X-ray contact microscopy (SXCM) is, at present, a useful tool for the examination at submicrometre resolution of biological systems maintained in their natural hydrated conditions. Among current X-ray-generating devices, laser-plasma sources are now easily available and, owing to their pulse nature, offer the opportunity to observe living biological samples before radiation damage occurs, even if the resolution achievable is not as high as with synchrotron-produced X-rays. To assess the potential of laser-plasma source SXCM in the study of cellular organelles, we applied it for the analysis of chloroplasts extracted from spinach leaves and mitochondria isolated from bovine heart and liver. X-ray radiation was generated by a nanosecond laser-plasma source, produced by a single shot excimer XeCl laser focused onto an yttrium target. The images obtained with SXCM were then compared with those produced by transmission electron microscopy observation of the same samples prepared with negative staining, a technique requiring no chemical fixation, in order to facilitate their interpretation and test the applicability of SXCM imaging.

Morphofunctional mitochondrial response to methylglyoxal toxicity in Bufo bufo embryos.

Methylglyoxal (2-oxopropanal) is a reactive alpha-oxoaldehyde that can be formed endogenously mainly as a by-product of glycolytic pathway. It is a cytotoxic compound with significant antiproliferative properties as it can bind, under physiological conditions, to nucleic acids and proteins, forming stable adducts. We have recently shown that exogenous methylglyoxal (150-600 microM) is highly toxic for amphibian embryos where it produces, when added to the culture water, inhibition of cell proliferation in the early developmental stages, followed by severe malformations and strongly reduced embryonic viability. In this work we investigate the morphofunctional effect of methylglyoxal on the common toad B. bufo embryo mitochondria in order to verify if its dysmorphogenetic action might be also ascribed to impairment of mitochondrial functions. The mitochondria were isolated from embryos at the developmental stages of morula, neural plate and operculum complete and developing in the presence of 600 microM methylglyoxal. The results show that exogenous methylglyoxal is highly toxic at mitochondrial level, where it produces proliferation, swelling and membrane derangement. As a consequence, mitochondria from treated embryos show decreased oxidative phosphorylation efficiency, as indicated by the significant reduction both of the respiratory control index values and of the embryonic ATP content. On the basis of these data, it is possible that the methylglyoxal-induced embryonic malformations as well as the strongly reduced viability might be also ascribed to energy depletion.

Developmental expression and distribution of amphibian glutathione transferases.

This work is aimed at detecting the expression and location of embryonic Bufo bufo GST (bbGSTP1-1) and adult B. bufo GST (bbGSTP2-2) during toad development, in order to assign a putative role to these enzymes also on the basis of their compartmentalization and to verify whether during the premetamorphic liver ontogeny the bbGSTP2-2 form appears. This study was also performed in the adult liver (the primary site of Pi class GST expression) and in the mature ovary, to discern if the embryonic form derives from maternal form. The results show that the embryos and the ovary express only bbGSTP1-1. Moreover, bbGSTP1-1 distribution is the same both in the early embryos and in the ovary: this strongly suggests that bbGSTP1-1 is of maternal origin. As development goes on, a wide distribution of bbGSTP1-1 all over the differentiating organs is observed. The embryonic liver expresses exclusively the bbGSTP1-1 form, while the adult liver is highly positive only towards the bbGSTP2-2 form. This implies that the switch towards the adult bbGSTP2-2 form occurs in metamorphic or postmetamorphic phases and that the detoxication metabolic requirements of the embryo may be completely fulfilled by the bbGSTP1-1 isoenzyme.

Biochemical, electrophoretic and immunohistochemical aspects of malate dehydrogenase in truffles (Ascomycotina).

The malate dehydrogenase (MDH; EC 1.1.1.37; L-malate-NAD(+)-oxidoreductase) activities of truffles of the genus Tuber (Tuber melanosporum Vittad., Tuber brumale Vittad., Tuber aestivum Vittad., Tuber magnatum Pico, Tuber rufum Pico) have been characterized with regard to the K(m) and V(max) values in the direct and reverse reactions. The isoelectrofocusing has revealed bands showing pI values ranging from pH 5.85 to 7.8. The MDH of T. melanosporum has been partially purified by hydroxyapatite treatment, DEAE-cellulose and Sephadex G-75 columns. With the partially purified T. melanosporum MDH activity polyclonal anti-T. melanosporum MDH antibodies have been prepared and used to localize MDH in the mycorrhizae and ascocarps of T. melanosporum. These antibodies inhibit T. melanosporum MDH activity as well as that of T. magnatum but not that of rabbit liver; this supports the specificity of the MDH antibodies used to localize MDH in truffle tissues.

Biochemical and ultrastructural alterations in rat after hyperoxic treatment: effect of taurine and hypotaurine.

The cell ultrastructure and some detoxifying enzyme activities were studied in skeletal muscles of young rats kept for 84 h under normobaric hyperoxia (95% O2) or normoxia as control. Rat were injected i.p.. Every 12 h either with 1 ml saline, 1 ml saline+30 mg hypotaurine or 1 ml saline+30 mg taurine. Ultrastructural observation revealed an highly protective effect on tissue damages due to hyperoxia in taurine-treated rats and, at less extent, in hypotaurine-treated ones. Enzymatic assays suggest a different mechanism of the two molecules in their protective action.

Age-dependent ultrastructural alterations and biochemical response of rat skeletal muscle after hypoxic or hyperoxic treatments.

This work deals with the antioxidant enzymatic response and the ultrastructural aspects of the skeletal muscle of young and aged rats kept under hypoxic or hyperoxic normobaric conditions. It is in fact well known that the supply of oxygen at concentrations higher or lower than those occurring under normal conditions can promote oxidative processes that can cause tissue damage. The enzymes investigated were both those directly involved in reactive oxygen species (ROS) scavenging (superoxide dismutase, catalase and selenium-dependent glutathione peroxidase), and those challenged with the detoxication of cytotoxic compounds produced by the action of ROS on biological molecules (glutathione transferase, glyoxalase I, glutathione reductase), in order to obtain a comparative view of the defence strategies used with respect to aging. Our results support the hypothesis that one of the major contributors to the aging process is the oxidative damage produced at least in part by an impairment of the antioxidant enzymatic system. This makes the aged organism particularly susceptible to oxidative stress injury and to the related degenerative diseases, especially in those tissues with high demand for oxidative metabolism.

Effect of methylglyoxal on Bufo bufo embryo development: morphological and biochemical aspects.

Methylglyoxal (2-oxopropanal) is a cytotoxic compound that can be formed endogenously as a by-product of glycolytic pathway; so its concentration is expected to increase when glycolysis activity increases such as during embryo development. In this work we study the effect of exogenous methylglyoxal on development and embryo viability during Bufo Bufo development and on the enzymes and cofactors involved in its detoxication process (glyoxalase I and II, reduced glutathione and glyceraldehyde 3-phosphate dehydrogenase). The results show that exogenous methylglyoxal does not affect the enzymatic pattern until stage 20, while it induces a significant activity decrease of the tested enzymes at stage 25. On the contrary methylglyoxal positively influences the reduced glutathione concentration at all the considered stages. At morphological and histological levels methylglyoxal causes a strong retardation of cell division in the early stages, that results in various abnormalities in the late development. In conclusion, methylglyoxal enters the embryo and is antiproliferative and teratogenic: the data further supports the hypothesis of the importance of the glyoxalase system in the process of cell growth and division.

Adaptive response of human melanoma cells to methylglyoxal injury.

The effects of methylglyoxal on the growth of a line of human melanoma cells are investigated. Methylglyoxal inhibits cell growth in a dose-dependent manner and causes an increase in glyceraldehyde 3-phosphate dehydrogenase, and glyoxalase 1 and glyoxalase 2 specific activities. The cellular response to increasing concentrations of methylglyoxal in the culture medium is also studied by measuring L-lactate production, reduced-oxidized glutathione levels and apoptotic cell death. Methylglyoxal seems to promote a change of cell population phenotypic repertoire toward a more monomorphic phenotype. In conclusion, methylglyoxal seems to induce an enzymatic cellular response that lowers methylglyoxal levels and selects the most resistant cells.

Melanogenesis, tyrosinase expression, and reproductive differentiation in black and white truffles (Ascomycotina).

White and black truffles of the genus Tuber are Ascomycotina as well as Neurospora crassa, which expresses tyrosinase dependently on the reproductive cycle. Tyrosinase expression dependent on reproductive differentiation has been also described in black truffles. We present novel and comparative work on melanogenic activities in black and white truffles that both express true tyrosinases. L-tyrosine 3-monooxygenase and L-DOPA oxidase activities colocalize as histochemically detected and are similarly located in white and black truffles, from the hypothecium through the sporogenic hyphae to asci and spores. Sulfur components of truffle flavours reversibly inhibit tyrosinase. The respiratory phenotype of truffle mitochondria is discussed in relation to reproductive differentiation and melanogenesis.

Molecular approach to the nucleo-melanosomal interaction in human melanoma cells.

This paper presents evidence that L-tyrosine oxidation products and 5,6-dihydroxyindole, an intermediate of melanin synthesis bind to and modify DNA structure, as tested by extracting cell DNA, using topoisomerase I and denaturation assays. When supercoiled plasmid pCU18 or pBR322 DNAs are treated with 5,6-dihydroxyindole the supercoiled species disappear and are converted to species less mobile in a gel retardation test with respect to relaxed DNA, 5,6-Dihydroxyindole causes an easier acid denaturation of the double helix. The results, that are dose dependent, would point to both intercalation and cross-linking of DNA by 5,6-dihydroxyindole and its oxidation product(s). 3H-L-tyrosine deriving radioactivity, bound to nuclear DNA, is higher at low pH, (5.6) if compared to pH 6.8. The highest radioactivity bound to cell DNA is found during the transition from the amelanotic to the melanotic phenotype in human melanoma cell lines. As a control, the binding of 3H-L-tyrosine radioactivity to human prostate fibroblast DNA was investigated.

Cyto-genotoxic species leakage within human melanoma melanosomes. Molecular-morphological correlations.

This work studies the phenotype changes, relating to pigment expression, of a human melanoma cell line. The phenotypic instabilities and proliferation rates are correlated with the production and release in the cell culture medium of active oxygen species and melanin synthesis intermediates. The proliferation rates versus L-tyrosine concentration in the culture media are investigated: a decrease is found when high L-tyrosine is added to the medium. This would be consistent with the release of cytotoxic and/or genotoxic species by melanoma cells. The morphology of melanoma melanosomes is coherent with the leakage of cytotoxic and genotoxic species produced during melanin synthesis.

Truffle melanogenesis: correlation with reproductive differentiation and ascocarp ripening.

The present work uses histochemical techniques to investigate the correlation between reproductive differentiation and age of truffles (Tuber aestivum and Tuber melanosporum) with melanin synthesis. The dopa oxidase and tyrosine hydroxylase activities of tyrosinase have been localized within the ascocarp and melanin localization was performed by the Schmorl's reaction. A true tyrosinase is present in truffles, able to oxidize both l-tyrosine and l-dopa. The tyrosinase activity is on in the young ascocarps (in the peridium, hypothecium, and fertile veins) and off in the ripe ones, thus it appears correlated with the age and differentiation of the sporogenic hyphae that arise from the hypothecium; a similar correlation has been previously described in Neurospora crassa, which is an ascomycete as well as the truffles.

Copper deficiency and pigmentation in the rat: morphofunctional aspects.

The effects of a low copper diet on pigmentation, pigment cell and melanosome morphology have been investigated in ACI/T male rats. After a three months treatment the fur and skin pigmentation is reduced as compared to the controls. The melanocytes of the treated rats show the phenotype of active pigment cells while some melanosomes are abnormally differentiated: both lamellar and granular organelles are present in the same pigment cell and mosaic age melanosomes appear. The abnormal melanosome structure expressed by the treated-rat melanocytes is also evident in vitro. After incubation with deoxycholate the melanosomes from the low-copper diet treated rats are much more altered than those from the control rats. The phenotype of the rats starved for copper seems to mimic as regards pigmentation the phenotype of the mouse Mo (mottled) mutation that is an experimental model of the Menkes' kinky hair syndrome. In conclusion copper deficiency seems to affect both the morphology and function of the pigment cells.

Liposome-entrapped tyrosinase: a tool to investigate the regulation of the Raper-Mason pathway.

The effect of the entrapment of mushroom tyrosinase (EC 1.14.18.1) within liposomes on the enzyme activity and Km vs. L-3,4-dihydroxyphenylalanine is reported in the present work; the effect of cholesterol insertion within liposome membranes on the enzyme activity has also been studied. The oxidation rates of various monophenols and diphenols by free and liposome-integrated mushroom tyrosinase were measured and the oxidation latencies vs. different substrates investigated. The different substrates are apparently oxidized according to the properties of the substituents as electron donors or acceptors; the Km values vs. L-3,4-dihydroxyphenylalanine calculated on measuring O2 consumption are higher than those calculated on measuring the dopachrome production rates. It is interesting that natural substrates of tyrosinase are oxidized according to a negative catalysis by the liposome-entrapped enzyme; this point is discussed in relation to the well known cytotoxicity of some intermediates of the Raper-Mason pathway.

A study on melanogenesis and catecholamine biosynthesis during Bufo bufo development.

Tyrosinase and L-DOPA decarboxylase activities have been investigated during Bufo bufo development since catecholamines and melanin are formed from common substrates in homologous cells. Catecholamines first appear at stage 13 (neural plate), but tyrosinase, at a very low level, and L-DOPA decarboxylase are present throughout all of prior development. Hence, L-DOPA decarboxylase activity is not likely to be correlated with the control of catecholamine synthesis, although at stage 17 it is mainly localized in the nonneural part of the embryo. The distribution of young melanosomes and L-DOPA decarboxylase suggest a separation between melanogenesis and catecholamine synthesis.

Developmental aspects of hexose metabolism in Bufo bufo.

Due to the close correlation between glucose mobilization and utilization within animal tissues, in this paper, the stages of appearance of phosphorylase, glucose-6-phosphatase and hexokinase as well as the levels of some intermediates of glucose metabolism have been investigated during Bufo bufo development. Phosphorylase first appears at stage 13 and is dominant in the neural part of the embryo, but, after this stage, increases relatively more in the nonneural one. Hexokinase appears at stage 17 and glucose-6-phosphatase soon after. Phosphorylase appearance at stage 13 is correlated with an increase of lactate content in the embryo; this may indicate a metabolization of hexoses. On this basis, the subsequent appearance of hexokinase and glucose-6-phosphatase activities also seems coherent with hexose mobilization and utilization within embryo. No direct causative factor for the changes observed was evident.

Some properties of Bufo bufo tyrosinase during development.

Tyrosinase expression during Bufo bufo development has been investigated. Until stage 19, only 1 electrophoretic band is detectable, but at a later stage (25) 3 bands appear. The Km for L-3,4-dihydroxyphenylalanine (L-dopa) was also determined.