Effect of growth factors and steroid hormones on heme oxygenase and cyclin D1 expression in primary astroglial cell cultures.

Astrocyte activity may be modulated by steroid hormones and GFs. This study investigates the interaction between glucocorticoids or estrogens and GFs on the expression of heme oxygenase-1 (HO-1) and cyclin D1 in astrocyte cultures at 14 days treated for 48 or 60 hr with dexamethasone (DEX) or 48 hr with 17ß-estradiol (E2) alone or with GFs added only in the last 12 or 24 hr. Twelve- or twenty-four-hour epidermal growth factor (EGF) treatment significantly enhanced HO-1 expression in astrocyte cultures pretreated for 48 hr with DEX. A highly significant increase in HO-1 expression was obtained after the last-12-hr EGF treatment in 48-hr E2-pretreated astrocyte cultures; this enhancement was particularly significant in 48-hr E2-pretreated cultures as well as in the last-12-hr insulin-treated ones pretreated for 48 hr with E2. Sixty-hour DEX-alone pretreatment as well as the last-12-hr EGF treatment in 60-hr DEX-pretreated astrocyte cultures showed a significant increase of cyclin D1 expression. A significant decrease of cyclin D1 expression in the last-12-hr insulin-like growth factor-I (IGF-1)-treated cultures pretreated for 60 hr with DEX was observed. A highly significant enhancement in cyclin D1 expression in 14 days in vitro astrocyte cultures pretreated with E2 alone for 48 hr and treated in the last 12 hr with IGF-1 in 48-hr E2-pretreated cultures was found. Finally, the data highlight an interactive dialogue between the growth factors and glucocorticoids or estrogens during the maturation of astroglial cells in culture that may control the HO-1 and cyclin D1 expression as well as proliferating astroglial cells during the cell cycle.

The critical role of didodecyldimethylammonium bromide on physico-chemical, technological and biological properties of NLC.

Exploiting the experimental factorial design and the potentiality of Turbiscan AG Station, we developed and characterized unmodified and DDAB-coated NLC prepared by a low energy organic solvent free phase inversion temperature technique. A 22 full factorial experimental design was developed in order to study the effects of two independent variables (DDAB and ferulic acid) and their interaction on mean particle size and zeta potential values. The factorial planning was validated by ANOVA analysis; the correspondence between the predicted values of size and zeta and those measured experimentally confirmed the validity of the design and the equation applied for its resolution. The DDAB-coated NLC were significantly affected in their physico-chemical properties by the presence of DDAB, as showed by the results of the experimental design. The coated NLC showed higher physical stability with no particles aggregation compared to the unmodified NLC, as demonstrated by Turbiscan(®) AGS measurements. X-ray diffraction, Raman spectroscopy and Cryo-TEM images allowed us to assert that DDAB plays a critical role in increasing the lipids structural order with a consequent enhancement of the NLC physical stability. Furthermore, the results of the in vitro biological studies allow the revisiting of the role of DDAB to the benefit of glioblastoma treatment, due to its efficacy in increasing the NLC uptake and reducing the viability of human glioblastoma cancer cells (U87MG).

Are pancreatic calcifications specific for the diagnosis of chronic pancreatitis? A multidetector-row CT analysis.

AIM: To retrospectively establish the most frequently encountered diagnoses in patients with pancreatic calcifications and to investigate whether the association of certain findings could be helpful for diagnosis. MATERIALS AND METHODS: One hundred and three patients were included in the study. The location and distribution of calcifications; presence, nature, and enhancement pattern of pancreatic lesions; pancreatic atrophy and ductal dilatation were recorded. Differences between patients with chronic pancreatitis and patients with other entities were compared by using Fisher's exact test. RESULTS: Patients had chronic pancreatitis (n=70), neuroendocrine tumours (n=14), intraductal papillary mucinous neoplasm (n=11), pancreatic adenocarcinoma (n=4), serous cystadenoma (n=4). Four CT findings had a specificity of over 60% for the diagnosis of chronic pancreatitis: parenchymal calcifications, intraductal calcifications, parenchymal atrophy, and cystic lesions. When at least two of these four criteria were used in combination, 54 of 70 (77%) patients with chronic pancreatitis could be identified, but only 17 of 33 (51%) patients with other diseases. When at least three of these four criteria were present, a specificity of 79% for the diagnosis of chronic pancreatitis was achieved. CONCLUSION: Certain findings are noted more often in chronic pancreatitis than in other pancreatic diseases. The presence of a combination of CT findings can suggest chronic pancreatitis and be helpful in diagnosis.

Effect of growth factors and steroids on transglutaminase activity and expression in primary astroglial cell cultures.

Type-2 transglutaminase (TG-2) is a multifunctional enzyme involved in the regulation of cell differentiation and survival that recently has been shown to play an emerging role in astrocytes, where it is involved in both proliferation and differentiation processes. Growth factors (GFs) such as EGF, basic fibroblast growth factor, insulin-like growth factor-I (IGF-I), and insulin (INS) are trophic and mitogenic peptides that participate in neuron-glia interactions and stimulate neuronal and astroglial proliferation and differentiation. Steroid hormones such as glucocorticoids and estrogens also play a pivotal role in neuronal and astroglial proliferation and differentiation and are key hormones in neurodegenerative and neuroprotective processes. We investigated the effects of the interaction of GFs with dexamethasone (DEX) or 17beta-estradiol (E(2)) on TG-2 activity and their expression in cultured astrocytes. We observed a significant increase in TG-2 activity and expression in astroglial cells treated for 24 hr with IGF-I, EGF, or INS. Priming of the cells with DEX or E(2), for 48 hr also led to an increase in TG-2 levels. When growth factors were present in the last 24 hr of the steroid treatment, a reduction in TG-2 expression and activity and a different subcellular TG-2 distribution were found. Our data indicate that steroid hormone-GF interaction may play an important role in astroglial function. The effect on TG-2 could be part of the regulation of intracellular pathways associated with the astrocyte response observed in physiological conditions and, possibly, also in neuropathological diseases.

Effects of cadmium exposure on testis apoptosis in the marine teleost Gobius niger.

It is known that heavy metals can accumulate in tissues during aquatic organism growth (bioaccumulation) and often biomagnify up the food chain interfering with the health and reproduction of both wildlife and humans. Recently, cadmium (Cd) was included in the endocrine disruptors list, exerting its effect on gametes quality and reproductive functions; in addition, its role as apoptotic factor was evidenced in different cell types and tissues. In the present study, the effects of two different Cd doses on testis and liver of the black goby Gobius niger were analyzed. Cd concentration in the water and its uptake by the gills were measured by differential pulse anodic stripping voltammetry. Toxic, apoptotic, and stressor Cd effects were analyzed using metallothionein (MTT), caspase 3 and heath shock protein 70 (HSP70), respectively, as bioindicators. The results of the present study suggested that, in the gills, the saturation of all specific metal sites was reached only with the highest Cd dose exposure. Either testis and liver showed an increase of MTT gene expression and protein synthesis in addition to HSP70 gene expression, related with Cd concentration in the water indicating that both tissues were affected by Cd exposure. In conclusion, the present study, not only shows the toxic effect of Cd on hepatic tissue, but also indicates its potency as apoptotic factor in the testis. This is supported by the increase of caspase 3 gene expression and the presence of its active form in testis of exposed fish.

Glutamate-evoked redox state alterations are involved in tissue transglutaminase upregulation in primary astrocyte cultures.

The aim of this study was to evaluate the involvement of oxidative stress in glutamate-evoked transglutaminase (TGase) upregulation in astrocyte cultures (14 DIV). A 24 h exposure to glutamate caused a dose-dependent depletion of glutathione intracellular content and increased the ROS production in cell cultures. These effects were receptor-mediated, as demonstrated by inhibition with GYKI 52466. The pre-incubation with glutathione ethyl ester or cysteamine recovered oxidative status and was effective in significantly reducing glutamate-increased tissue TGase. These data suggest that tissue TGase upregulation may be part of a biochemical response to oxidative stress induced by a prolonged exposure of astrocyte cultures to glutamate.

Cystamine inhibits transglutaminase and caspase-3 cleavage in glutamate-exposed astroglial cells.

Although the precise role of transglutaminase in cell death is unknown, several findings demonstrate that tissue transglutaminase selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Calcium-dependent transglutaminase reactions are also implicated in several neurodegenerative diseases, including alterations in the release of excitatory amino acids. One prevalent theme in cell damage induced by excitotoxic stimuli in different regions of the CNS is that apoptosis may be executed by intracellular caspase proteases. Furthermore, the presence of functional ion channel-gated receptors in glial cells suggests that also astrocytes can be susceptible to glutamate's toxic effects. In this study, we demonstrated that prolonged exposure to glutamate (100 microM) of cultured astrocytes caused an increase in the expression of tissue transglutaminase (tTG). This effect was prevented by preincubation with GYKI 52466, an antagonist of AMPA/KA receptors. Glutamate exposure also promoted an increase in caspase-3 compared with control cultures. Confocal laser microscopy analysis demonstrated the presence of activated caspase-3 in the cytoplasm as well as in the nucleus. The inhibition of TG-catalyzed reactions by cystamine (1 mM) blocked the activation pathway of caspase-3, with an evident reduction of enzyme cleavage. These results suggest that glutamate increased both TG and caspase-3 in astroglial cells early in the excitotoxin-induced events.

[Locoregional chemotherapy of locally advanced breast cancer in a pre-treated patient: case report]. / Chemioterapia locoregionale nel carcinoma mammario localmente avanzato in paziente pre-trattata: case report.

We report of the use of intra-arterial chemotherapy in one case of locally advanced breast cancer, that had been systemically pre-treated. Locoregional chemotherapy was delivered via percutaneous access. The catheter tip was placed into the subclavian artery and into origin of the internal mammary artery; it was removed after every cycle of treatment. The schedule of chemotherapy was: epirubicin 30 mg/m2, mitomycin 7 mg/m2 and 5 fluouracil 1000 mg. Three cycles were administered, and the treatment was well tolerated. The patient responded to intra-arterial chemotherapy, and she subsequently underwent complete surgical resection. Intra-arterial chemotherapy for breast cancer in an uncommon approach to the treatment of locally advanced disease. Nevertheless, in selected cases, it could be a more effective therapeutic option for patients with systemic chemotherapy-resistant disease.

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.

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.

Effects of glutathione depletors on post-ischemic reperfusion in rat brain.

The present paper reports the effects of GSH depletion (diethylmaleate induced) on partial cerebral ischemia and reperfusion for 7 and 20 days. Our results confirm that there is a paradoxical protective effect of the GSH-depletor and suggest an improved neuronal trophism induced by diethylmaleate treatment.

Glutamine synthetase activity and HSP70 levels in cultured rat astrocytes: effect of 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine.

The ether lipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) is a membrane interactive drug selectively cytotoxic toward neoplastic cells compared to normal cells. It induces apoptosis in human leukemic HL-60, T-lymphoid and in U937 myeloid cell lines and stimulates NO biosynthesis in cultured rat astrocytes. We have found a double action of ET-18-OCH3 in astrocytes which, at low doses, promotes a moderate induction of heat shock proteins of 70 kDa (HSP70) and the increase of glutamine synthetase (GS) activity. Conversely, at high doses, the drug shows toxic effects on astrocytes inducing decrease in GS activity, low molecular weight DNA formation, and release of lactic dehydrogenase (LDH) in the culture medium. Its analog compound platelet-activating factor (PAF) shares some of these biological aspects.

Enhanced resistance of adriamycin-treated MCR-5 lung fibroblasts by increased intracellular glutathione peroxidase and extracellular antioxidants.

Considerable evidence indicates that reactive oxygen species play an etiological role in both cardiotoxicity and the skin necrosis induced by adriamycin (ADM). An increase in glutathione peroxidase activity on addition of selenium to cultured MCR-5 lung fibroblasts was observed; this increase was accompanied by enhanced cellular resistance to ADM toxicity. Moreover, the presence of exogenous antioxidant systems, such as superoxide dismutase, catalase, vitamin E, dimethylsulfoxide, and desferroxamine, an iron chelating agent, resulted in significant protection from ADM-mediated damage.

Free radical scavenger depletion in post-ischemic reperfusion brain damage.

In the present study the influence of pretreatment with various GSH depletors such as buthionine sulfoximine (BSO) and diethylmaleate (DEM) was investigated in rats following cerebral post-ischemic reperfusion. Moreover, the effect of diethyldithiocarbamic acid (DDC), inhibitor of endogenous Cu,Zn-SOD, was evaluated. A significant depletion (40% of control value) of GSH levels was observed 24 h after DEM administration; after 48 h the value reached control levels. BSO showed maximal GSH depletion (59%) 24 h after administration and it was constant for almost 48 h. DDC administration caused a marked decrease (60%) of Cu,Zn-SOD activity 4 h after the injection and induced a marked decrease in percentage of survival with respect to control (untreated, ischemic) rats, when administered 4 h before ischemia. BSO and DEM prolonged the survival time of animals when administered 24 h before ischemia. This last paradoxical effect is unclear at present, but it might be due to an influence on glutamate cascade.

Crossfostering and early development of natural killer cytotoxic activity in various inbred mouse strains.

In the present study using crossfostering among three inbred mouse strains (C57BL/6, DBA/2, and Balb/c) we compared the effects of lactation with milk of different compositions on the development of NK cells cytotoxic activity. The results show that the pups from C57BL/6 and DBA/2 mice exhibit a significant early increase of NK cells cytotoxic activity when fostered by Balb/c dams, in comparison to those fostered by natural mothers. The analysis of proteins, lactose, and lipids showed difference among the strains for all components. Strain effects for days of lactation were also observed. The naso-anal length and the body weight of young mice showed differences with the strain of fostering mothers. The results indicate that the characteristic of maternal milk composition interacts with the inbred genetic susceptibility of the pups to elicit the full expression of the level of NK activity.

Lipid peroxidation in rat cerebral cortex during post-ischemic reperfusion: effect of exogenous antioxidants and Ca(++)-antagonist drugs.

Although the role of oxidant-antioxidant metabolism in total ischemia and reperfusion in the central nervous system and cardiac myocardium have been well studied, less is known about the consequences of partial ischemic episodes. Here we show that reperfusion contributes to free radical formation as judged by conjugated diene formation. Also, antioxidants and Ca++ antagonists were able to reduce free radical formation. These results would suggest that free radical generation following ischemia and reperfusion may result from more than one injury process in cerebral cortex.

Superoxide dismutase activity and reduced glutathione content in cataractous lens of patients with glucose-6-phosphate dehydrogenase deficiency.

In the present research we have assayed the glucose-6-phosphate dehydrogenase, superoxide dismutase activities and reduced glutathione content in human cataractous lenses of 83 Sicilian subjects. Five of 45 males were G6PD deficient, whereas eight of 38 females showed a significant reduction in G6PD by 50%. The five males hemizygous for G6PD defect showed undetectable G6PD activity and low GSH levels in their lenses when compared to cataractous patients without erythrocyte G6PD deficiency; on the contrary, the specific activity of lenticular total SOD was found to be significantly increased. The G6PD and SOD activities as well as GSH levels, in the lenses of eight females with intermediate erythrocyte G6PD levels, were not significantly different when compared to females with normal erythrocyte G6PD activity.