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Gliomas are the most common primary brain tumor affecting human adults and remain a therapeutic challenge because cells of origin are still unknown. Here, we investigated the cellular origin of low-grade gliomas in a rat model based on transplacental exposure to Nethyl-N-nitrosourea (ENU). Longitudinal magnetic resonance imaging coupled to immunohistological and immunocytochemical analyses were used to further characterize low-grade rat gliomas at different stages of evolution. We showed that early low-grade gliomas have characteristics of oligodendroglioma-like tumors and exclusively contain NG2-expressing slow dividing precursor cells which express early markers of oligodendroglial lineage. These tumor-derived precursors failed to fully differentiate into oligodendrocytes and exhibited multipotential abilities in vitro. Moreover, a few glioma NG2+ cells are resistant to radiotherapy and may be responsible for tumor recurrence, frequently observed in humans. Overall, these findings suggest that transformed multipotent NG2 glial precursor cell may be a potential cell of origin in the genesis of rat ENU-induced oligodendroglioma-like tumors. This work may open up new perspectives for understanding biology of human gliomas.

The hypothesis that life span extension by caloric restriction (CR) is contingent upon the attenuation of macromolecular oxidative damage was tested in two different strains of mice: the C57BL/6, whose life span is extended by CR, and the DBA/2, in which CR has relatively minor or no impact on longevity. Mice were fed ad libitum (AL) or restricted to 40% lesser food, starting at 4 months of age. Protein damage was measured as protein-linked adducts of 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) in skeletal muscle mitochondria at 6- and 23-months of age. Protein-HNE and -MDA content increased with age in C57BL/6 mice and CR significantly attenuated these augmentations. Metalloprotease 1, NADP-dependent mitochondrial malic enzyme (isoform 2) and citrate synthase were identified by mass spectroscopy to contain HNE/MDA adducts. DBA/2 mice exhibited little effect of age or CR on protein HNE/MDA content in skeletal muscle mitochondria. In contrast, protein-HNE levels in liver mitochondria showed a significant increase with age in AL-fed mice of both strains, and CR caused significant attenuation of this damage. Overall, results indicated that the age-related increase in protein oxidative damage and its abatement by CR are genotype- and tissue- specific, and not a universal phenomenon.

Long-term culturing of brain cells from neonatal BD-IX rats after transplacental treatment with N-ethyl-N-nitrosourea (ENU) results in malignantly transformed cells after a lag period of about 250 days. During culturing, the brain cells undergo a sequence of morphological changes. We examined oncogene expression in cultured cells from ENU-treated animals and found that transformed glioma cells differ from premalignant glial cells by containing high levels of c-sis transcripts. We also report that the transformed cells synthesize functional platelet-derived growth factor. Because glial cells have receptors for platelet-derived growth factor, we propose that an autocrine mechanism plays an important role in ENU-induced brain tumorigenesis.

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Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53 percent of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28 and 30 percent, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46 percent, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16 percent). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.

We found that adenosine 5′-monophosphate-activated protein kinase (AMPK)—which is considered the “fuel sensor” of mammalian cells because it directly responds to the depletion of the fuel molecule ATP—is strongly activated by tumor-like hypoxia and glucose deprivation. We also observed abundant AMPK activity in tumor cells in vivo, using subcutaneous tumor xenografts prepared from cells transformed with oncogenic H-Ras. Such rapidly growing transplants of tumor cells, however, represent fully developed tumors that naturally contain energetically stressed microenvironments that can activate AMPK. Therefore, to investigate the induction of AMPK activity during experimental tumorigenesis, we used an established model of brain tumor (glioma) development in the offspring of rats exposed prenatally to the mutagen N-ethyl-N-nitrosourea (ENU). We observed that immunostaining for a specific readout of AMPK activity (AMPK-dependent phosphorylation of acetyl-CoA carboxylase) was prominent during ENU-initiated neurocarcinogenesis, from the occurrence of early hyperplasia (microtumors) to the emergence of large gliomas. Moreover, we observed that immunostaining for activating phosphorylation of AMPK correlated with the same stages of glioma development, notably in mitotic tumor cells where the signal showed punctate as well as cytoplasmic patterns associated with spindle formation. Based on these observations, we propose that neurocarcinogenesis requires AMPK-dependent regulation of cellular energy metabolism.

The cyclin-dependent kinase (CDK) inhibitor p16INK4a and the MDM2 ubiquitin ligase inhibitor p19ARF are critical to the regulation of cell cycle progression. Their loss by deletion, mutation or epigenetic silencing is a common molecular alteration in many human cancers. To investigate the role of p16INK4a/p19ARF deficiency in CNS tumor pathogenesis, pregnant mice bearing p16-/-/p19-/-, p16+/-/p19+/-, and p16+/+/p19+/+ embryos were exposed transplacentally on gestation day 14 to a single dose of the potent carcinogen, ethylnitrosourea (ENU). p16+/-/p19+/- male mice treated with ENU developed meningial proliferative lesions with a high incidence (5/10). The incidence was lower in other ENU-treated animals of both sexes and none occurred in saline-treated control animals. The lesions ranged from widespread meningeal proliferation and plaque-like thickening by neoplastic spindle cells consistent with meningiomatosis to a larger discrete mass consistent with a meningioma. Ultrastructural analysis revealed the presence of intercellular junctions between cells, supporting a meningothelial histogenesis. Spontaneous meningiomas occur rarely in wild-type mice but are a common neoplasm afflicting humans, accounting for between 13 and 26% of primary intracranial neoplasms. This ENU inducible meningeal lesion in p16+/-/p19+/- mice may provide additional insight into the pathogenesis of meningeal neoplasia and aid the development of therapeutics.

Gliomas are tumors of glial origin formed in the central nervous system and exhibit profound morphological and genetic heterogeneity. The etiology of this heterogeneity involves an interaction between genetic alterations and environmental risk factors. Scientific evidence suggests that certain natural dietary components, such as phytoestrogens, flavonoids, polyunsaturated fatty acids and vitamins may exert a protective effect against gliomas by changing the nature of the interaction between genetics and environment. Similarly, certain anti-inflammatory drugs and dietary modifications, such as methionine restriction and the adoption of low-calorie or ketogenic diets, may take advantage of glioma and normal glial cells’ differential requirements for glucose, methionine, and ketone bodies and may therefore be effective as part of preventive or treatment strategies for gliomas. Treatment trials of glioma patients and chemoprevention trials of individuals with a known genetic predisposition to glioma using the most promising of these agents, such as the anti-inflammatory drugs curcumin and gamma-linolenic acid, are needed to validate or refute these agents’ putative role in gliomas.

Calorie restriction extends lifespan by decreasing the rate of tumor formation, an effect occurring within 8 weeks of initiating a restricted diet. Our goal was to define how the first weeks of a calorie restricted diet (60% of ad libitum calories) affects putative mediators of the calorie restriction phenotype, focusing on regulators of fatty acid biosynthesis. In C57Bl/6 mice, insulin decreased over 50% (p<0.05) during the first week of calorie restriction whereas IGF-1 was unaffected. In the liver, PPAR mRNA fell to 13% of baseline after one week of calorie restriction (p<0.05), whereas hepatic SREBP-1c and SIRT1 mRNA levels were unaffected. No changes in abdominal or subcutaneous adipose tissue were observed until after 8 weeks of caloric restriction. We conclude that calorie restriction-induced decreases in insulin and hepatic PPAR are rapid enough to support a role for these molecules in triggering the initial phase of the calorie restriction phenotype.

COPD is associated with an increased load on the diaphragm. Since chronic muscle loading results in changes in antioxidant capacity and formation of reactive oxygen and reactive nitrogen species, we hypothesized that COPD has a similar effect on the diaphragm, which is related to the severity of COPD. Catalase activity was determined spectrophotometrically. Levels of 4-hydroxy-2-nonenal (HNE)-protein adducts and 3-nitrotyrosine (NT) formation were measured using western blotting. Levels of malondialdehyde (MDA) were assessed by high-performance liquid chromatography. We found that catalase activity was ~89% higher in the diaphragm of severe COPD patients (FEV1 37 ± 5% predicted) compared with non-COPD patients. MDA levels, a marker for lipid peroxidation, were significantly lower in the diaphragm of COPD patients compared with non-COPD patients, whereas the level of HNE-protein adducts was equal in both groups. NT formation was not different between groups. However, increasing hyperinflation and NT formation were inversely correlated. These results indicate that in COPD the diaphragm adapts to a higher work load by increasing catalase activity, resulting in a reduction in oxidative damage to lipids and tyrosine nitration of proteins.

Summary

To determine if short-term calorie restriction reverses vascular endothelial dysfunction in old mice, old (O, n=30) and young (Y, n=10) male B6D2F1 mice were fed ad libitum (AL) or calorie restricted (CR, ∼30%) for 8 weeks. Ex vivo carotid artery endothelium-dependent dilation (EDD) was impaired in OAL vs. YAL (74±5 vs. 95±2% of maximum dilation, P<0.05), whereas OCR and YCR did not differ (96±1 vs. 94±3%). Impaired EDD in OAL was mediated by reduced nitric oxide (NO) bioavailability associated with decreased endothelial NO synthase expression (aorta) (P<0.05), both of which were restored in OCR. Nitrotyrosine, a cellular marker of oxidant modification, was markedly elevated in OAL (P<0.05), whereas OCR was similar to Y. Aortic superoxide production was 150% greater in OAL vs. YAL (P<0.05), but normalized in OCR, and TEMPOL, a superoxide dismutase (SOD) mimetic that restored EDD in OAL (to 97±2%), had no effect in Y or OCR. OAL had increased expression and activity of the oxidant enzyme, NADPH oxidase, and its inhibition (apocynin) improved EDD, whereas NADPH oxidase in OCR was similar to Y. Manganese SOD activity and sirtuin1 expression were reduced in OAL (P<0.05), but restored to Y in OCR. Inflammatory cytokines were greater in OAL vs. YAL (P<0.05), but unaffected by CR. Carotid artery endothelium-independent dilation did not differ among groups. Short-term CR initiated in old age reverses age-associated vascular endothelial dysfunction by restoring NO bioavailability and reducing oxidation stress via reduced NADPH oxidase-mediated superoxide production and stimulation of anti-oxidant enzyme activity, and upregulates sirtuin1.

Background

Accumulating data suggest that liver is a major target organ of systemic effects observed in the presence of a cancer. In this study, we investigated the consequences of the presence of chemically induced brain tumors in rats on biophysical parameters accounting for the dynamics of water in liver mitochondria.

Methods

Tumors of the central nervous system were induced by intraveinous administration of ethylnitrosourea (ENU) to pregnant females on the 19th day of gestation. The mitochondrial crude fraction was isolated from the liver of each animal and the dynamic parameters of total water and its macromolecule-associated fraction (structured water, H2Ost) were calculated from Nuclear Magnetic Resonance (NMR) measurements.

Results

The presence of a malignant brain tumor induced a loss of water structural order that implicated changes in the physical properties of the hydration shells of liver mitochondria macromolecules. This feature was linked to an increase in the membrane cholesterol content, a way to limit water penetration into the bilayer and then to reduce membrane permeability. As expected, these alterations in mitochondrial plasticity affected ionic exchanges and led to abnormal features of mitochondrial biogenesis and caspase activation.

Conclusion

This study enlightens the sensitivity of the structured water phase in the liver mitochondria machinery to external conditions such as tumor development at a distant site. The profound metabolic and functional changes led to abnormal features of ion transport, mitochondrial biogenesis and caspase activation.

Objective

Free radical-mediated reactions have been implicated as contributors in a number of autoimmune disease (ADs) including SLE. However, potential of oxidative/nitrosative stress in eliciting an autoimmune response, or in disease prognosis and pathogenesis in humans remains largely unexplored. This study investigates the status and contribution of oxidative/nitrosative stress in SLE.

Methods

Sera from 72 SLE patients with various SLE scores [SLE disease activity index (SLEDAI)] and 36 age- and gender-matched controls were evaluated for oxidative/nitrosative stress markers, such as anti-malondialdehyde (MDA)- and anti-4-hydroxynonenal (HNE)-protein adduct antibodies, MDA-/HNE-protein adducts, superoxide dismutase (SOD), nitrotyrosine and iNOS.

Results

Serum analysis showed significantly higher levels of both anti-MDA-/anti-HNE-protein adduct antibodies and MDA-/HNE-protein adducts in SLE patients. Interestingly, our data showed not only increased number of subjects positive for anti-MDA- or anti-HNE-protein antibodies, but also greater increases in both these antibodies in SLE patients with greater SLEDAI (≥ 6), which were significantly higher than the lower SLEDAI group (<6). Data also showed a significant correlation between anti-MDA or anti-HNE antibodies and SLEDAI (r = 0.734 and 0.647 for anti-MDA and anti-HNE antibodies, respectively) suggesting a possible causal relationship between these antibodies and SLE. Furthermore, sera from SLE patients had lower levels of SOD, and higher levels of iNOS and nitrotyrosine.

Conclusion

Our findings support an association between oxidative/nitrosative stress and SLE. Stronger response in samples with higher SLEDAI suggests that oxidative/nitrosative stress markers may be useful in evaluating the progression of SLE as well as in elucidating the mechanisms of disease pathogenesis.

A dysregulation of the redox homoeostasis has been reported in various neoplastic disorders. Malondialdehyde/4-hydroxy-2,3-nonenal (MDA/HNE) and protein carbonyl groups represent in vivo indexes of lipid peroxidation and protein oxidation, respectively, suitable to investigate radical-mediated physio-pathological conditions. We evaluated MDA/HNE and protein carbonyl groups in sera of untreated Hodgkin's lymphoma (HL) patients in advanced disease stages, in order to quantify the oxidative stress. HL patients displayed significantly higher levels of both MDA/HNE and protein carbonyl groups as compared with healthy controls. This is the first evidence that a strong increase in HL is one of the most common haematological malignancies, representing approximately 30% of all lymphomas in the circulating protein carbonyl content in HL. These findings may contribute to a better definition of the redox homoeostasis dysregulation in HL.

Doxorubicin (Dox) is a potent, broad-spectrum chemotherapeutic drug used around the world. Despite its effectiveness, it has a wide range of toxic side effects, many of which most likely result from its inherent pro-oxidant activity. It has been reported that Dox has toxic effects on normal tissues, including brain tissue. The present study tested the protective effect of a xanthone derivative of Garcinia Mangostana against Dox-induced neuronal toxicity. Xanthone can prevent Dox from causing mononuclear cells to increase the level of tumor necrosis factor-alpha (TNFα). We show that xanthone given to mice before Dox administration suppresses protein carbonyl, nitrotyrosine and 4-hydroxy-2′-nonenal (4HNE)-adducted proteins in brain tissue. The levels of the pro-apoptotic proteins p53 and Bax and the anti-apoptotic protein Bcl-xL were significantly increased in Dox-treated mice compared with the control group. Consistent with the increase of apoptotic markers, the levels of caspase-3 activity and TUNEL-positive cells were also increased in Dox-treated mice. Pretreatment with xanthone suppressed Dox-induced increases in all indicators of injury tested. Together, the results suggest that xanthone prevents Dox-induced central nervous system toxicity, at least in part, by suppression of Dox-mediated increases in circulating TNFα. Thus, xanthone is a good candidate for prevention of systemic effects resulting from reactive oxygen generating anticancer therapeutics.

Identification of biological mediators in sarcopenia is pertinent to the development of targeted interventions to alleviate this condition. Iron is recognized as a potent pro-oxidant and a catalyst for the formation of reactive oxygen species in biological systems. It is well accepted that iron accumulates with senescence in several organs, but little is known about iron accumulation in muscle and how it may affect muscle function. In addition, it is unclear if interventions which reduced age-related loss of muscle quality, such as calorie restriction, impact iron accumulation. We investigated non-heme iron concentration, oxidative stress to nucleic acids in gastrocnemius muscle and key indices of sarcopenia (muscle mass and grip strength) in male Fischer 344 X Brown Norway rats fed ad libitum (AL) or a calorie restricted diet (60% of ad libitum food intake starting at 4 months of age) at 8, 18, 29 and 37 months of age. Total non-heme iron levels in the gastrocnemius muscle of AL rats increased progressively with age. Between 29 and 37 months of age, the non-heme iron concentration increased by approximately 200% in AL-fed rats. Most importantly, the levels of oxidized RNA in gastrocnemius muscle of AL rats were significantly increased as well. The striking age-associated increase in non-heme iron and oxidized RNA levels and decrease in sarcopenia indices were all attenuated in the calorie restriction (CR) rats. These findings strongly suggest that the age-related iron accumulation in muscle contributes to increased oxidative damage and sarcopenia, and that CR effectively attenuates these negative effects.

Bone mass is correlated with body weight during growth. However, it is unclear how bone mass is influenced by weight gain following skeletal maturity. The purpose of this study was to determine the effects of weight maintenance and two rates of weight gain on bone metabolism using skeletally mature female rats. Eight-month-old female rats were fed one of 3 diets for 13 weeks: Lieber DeCarli liquid diet ad libitum (control diet), the same diet with caloric restriction to maintain initial body weight (calorie-restricted diet), and the same diet fed ad lib with the exception that appetite was enhanced (calorie-increased diet) by replacing a small quantity of maltose-dextran isolcalorically with ethanol (0.5% caloric intake). Compared to baseline, rats fed the calorie-restricted, control, and calorie-increased diets changed in weight by −1 ± 2% (mean ± SE), 10 ± 3%, and 21 ± 2%, respectively. Weight gain was associated with a significant increase in serum leptin, a putative regulator of bone formation. In contrast, significant differences in tibial bone mineral content and density were not detected among treatments groups following dietary intervention or between treatment groups and the baseline group. Similarly, indices of cancellous bone architecture (area, trabecular number, thickness, and separation) and bone turnover (mineralizing perimeter, mineral apposition rate, and bone formation rate) did not differ among groups following dietary intervention. Our findings suggest that neither weight gain nor increased serum leptin levels, over the range evaluated, influence bone metabolism in skeletally mature female rats.

Aging is associated with protein damage and imbalance in redox status in a variety of cells and tissues, yet little is known about the extent of age-related oxidative stress in the peripheral nervous system. Previously, we showed a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. The age-related decline in functional molecules is associated with alterations in cellular protein homeostatic mechanisms, which could lead to a buildup of damaged, aggregated proteins. To determine the extent of oxidative damage within myelinated peripheral nerves, we studied sciatic nerves from rats of four different ages (8, 18, 29, and 38 months) maintained on an ad libitum or a 40% calorie-restricted diet. We found a prominent accumulation of polyubiquitinated substrates with age, which are associated with the conglomeration of distended lysosomes and lipofuscin adducts. The occurrence of these structures is notably less frequent within nerves of age-matched rodents kept on a lifelong reduced calorie diet. Markers for lipid peroxidation, inflammation, and immune cell infiltration are all elevated in nerves of ad libitum–fed rats, whereas food restriction is able to attenuate such deleterious processes with age. Together these results show that dietary restriction is an efficient means of defying age-related oxidative damage and maintaining a younger state in peripheral nerves.

Abstract

Aging is associated with protein damage and imbalance in redox status in a variety of cells and tissues, yet little is known about the extent of age-related oxidative stress in the peripheral nervous system. Previously, we showed a drastic decline in the expression of glial and neuronal proteins in myelinated peripheral nerves with age, which is significantly ameliorated by lifelong calorie restriction. The age-related decline in functional molecules is associated with alterations in cellular protein homeostatic mechanisms, which could lead to a buildup of damaged, aggregated proteins. To determine the extent of oxidative damage within myelinated peripheral nerves, we studied sciatic nerves from rats of four different ages (8, 18, 29, and 38 months) maintained on an ad libitum or a 40% calorie-restricted diet. We found a prominent accumulation of polyubiquitinated substrates with age, which are associated with the conglomeration of distended lysosomes and lipofuscin adducts. The occurrence of these structures is notably less frequent within nerves of age-matched rodents kept on a lifelong reduced calorie diet. Markers for lipid peroxidation, inflammation, and immune cell infiltration are all elevated in nerves of ad libitum–fed rats, whereas food restriction is able to attenuate such deleterious processes with age. Together these results show that dietary restriction is an efficient means of defying age-related oxidative damage and maintaining a younger state in peripheral nerves.

Background

Even in the presence of oxygen, malignant cells often highly depend on glycolysis for energy generation, a phenomenon known as the Warburg effect. One strategy targeting this metabolic phenotype is glucose restriction by administration of a high-fat, low-carbohydrate (ketogenic) diet. Under these conditions, ketone bodies are generated serving as an important energy source at least for non-transformed cells.

Methods

To investigate whether a ketogenic diet might selectively impair energy metabolism in tumor cells, we characterized in vitro effects of the principle ketone body 3-hydroxybutyrate in rat hippocampal neurons and five glioma cell lines. In vivo, a non-calorie-restricted ketogenic diet was examined in an orthotopic xenograft glioma mouse model.

Results

The ketone body metabolizing enzymes 3-hydroxybutyrate dehydrogenase 1 and 2 (BDH1 and 2), 3-oxoacid-CoA transferase 1 (OXCT1) and acetyl-CoA acetyltransferase 1 (ACAT1) were expressed at the mRNA and protein level in all glioma cell lines. However, no activation of the hypoxia-inducible factor-1α (HIF-1α) pathway was observed in glioma cells, consistent with the absence of substantial 3-hydroxybutyrate metabolism and subsequent accumulation of succinate. Further, 3-hydroxybutyrate rescued hippocampal neurons from glucose withdrawal-induced cell death but did not protect glioma cell lines. In hypoxia, mRNA expression of OXCT1, ACAT1, BDH1 and 2 was downregulated. In vivo, the ketogenic diet led to a robust increase of blood 3-hydroxybutyrate, but did not alter blood glucose levels or improve survival.

Conclusion

In summary, glioma cells are incapable of compensating for glucose restriction by metabolizing ketone bodies in vitro, suggesting a potential disadvantage of tumor cells compared to normal cells under a carbohydrate-restricted ketogenic diet. Further investigations are necessary to identify co-treatment modalities, e.g. glycolysis inhibitors or antiangiogenic agents that efficiently target non-oxidative pathways.

The anticarcinogenic effects and mechanisms of the biotechnological drugs of Panax ginseng C.A. Meyer cultivated in Russia, bioginseng, panaxel and panaxel- 5, were studied. Bioginseng was produced from a tissue culture of ginseng root cultured on standard medium, whereas panaxel and panaxel-5 were produced from ginseng tissue root cultures using standard mediums enriched with 2-carboxyethylgermanium sesquioxide and 1-hydroxygermatran-monohydrate respectively. All three ginseng drugs inhibited the development of mammary tumors induced by intramammary injections of N-methyl-N-nitrosourea (MNU) in rats, the development of the brain and spinal cord tumors induced by transplacental administration of N-ethyl-N-nitrosourea (ENU) in rats, and the development of uterine, cervical and vaginal tumors induced by intravaginal applications of 7,12-dimethylbenz(a)anthracene (DMBA) in mice. The ginseng drugs induced the cytotoxic activity of macrophages in mice, enhanced T-lymphocyte rosette formation in guinea pigs exposed to cyclophosphamide, and stimulated the production of thyroid hormones in rats. These mechanisms may contribute to the anticarcinogenic action of the ginseng drugs. The organic germanium compounds present in panaxel and panaxel-5 did not potentiate the anticarcinogenic or immuno- stimulatory effects as much as biogeinseng. Preliminary clinical trials with panaxel and bioginseng were carried out in patients with precancerous lesions of the esophagus and endometrium. Panaxel was found to have a strong therapeutic effect in patients suffering from chronic erosive esophagitis. Bioginseng induced the regression of adenomatous-cystic hyperplasia of the endometrium in some patients. Thus, we conclude that the drugs of ginseng appear to hold considerable promise for future cancer chemoprevention.

Oxidative stress has been shown to underlie neuropathological aspects of Alzheimer's disease (AD). 4-Hydroxy-2-nonenal (HNE) is a highly reactive product of lipid peroxidation of unsaturated lipids. HNE-induced oxidative toxicity is a well-described model of oxidative stress-induced neurodegeneration. GSH plays a key role in antioxidant defense, and HNE exposure causes an initial depletion of GSH that leads to gradual toxic accumulation of reactive oxygen species. In the current study, we investigated whether pre-treatment of cortical neurons with acetyl-L-carnitine (ALCAR) and α-lipoic acid (LA) plays a protective role in cortical neuronal cells against HNE-mediated oxidative stress and neurotoxicity. Decreased cell survival of neurons treated with HNE correlated with increased protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (HNE) accumulation. Pretreatment of primary cortical neuronal cultures with ALCAR and LA significantly attenuated HNE-induced cytotoxicity, protein oxidation, lipid peroxidation and apoptosis in a dose-dependent manner. Additionally, pretreatment of ALCAR and LA also led to elevated cellular GSH and heat shock proteins (HSPs) levels compared to untreated control cells. We have also determined that pretreatment of neurons with ALCAR and LA leads to the activation of phosphoinositol-3 kinase (PI3K), PKG and ERK1/2 pathways, which play essential roles in neuronal cell survival. Thus, this study demonstrates a cross talk between the PI3K, PKG and ERK1/2 pathways in cortical neuronal cultures that contributes to ALCAR and LA-mediated pro-survival signaling mechanisms. This evidence supports the pharmacological potential of co-treatment of ALCAR and LA in the management of neurodegenerative disorders associated with HNE-induced oxidative stress and neurotoxicity, including AD.

BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.

4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation (LPO) product formed during oxidative stress. 4-HNE is highly reactive toward cellular nucleophiles and is implicated in the evolution of numerous pathologies associated with oxidative stress and LPO. Recent evidence suggests that chronic pro-oxidant exposure results in the loss of extracellular signal-regulated kinase (Erk)-1/2 phosphorylation in vivo, a signaling pathway associated with cellular proliferation, survival, and homeostasis. Immunodetection and molecular analysis were used in this study to evaluate the hypothesis that 4-HNE modification of Erk-1/2 inhibits constitutive Erk-Est-like protein (Elk)-1-activating protein (AP)-1 signaling. Primary rat hepatocytes treated with subcytotoxic, pathologically relevant concentrations of 4-HNE demonstrated a concentration-dependent loss of constitutive Erk-1/2 phosphorylation, activity, and nuclear localization. These findings were consistent with iron-induced intracellular LPO, which also resulted in a concentration-dependent decrease in hepatocyte Erk-1/2 phosphorylation and activity. 4-HNE and iron-induced inhibition of Erk-1/2 was inversely correlated with the accumulation of 4-HNE-Erk-1/2 monomer adducts. 4-HNE treatment of hepatocytes decreased nuclear total and phosphorylated Erk-1/2, Elk-1, and AP-1 phosphorylation as well as cFos and cJun activities. The cytosolic modification of unphosphorylated Erk-1/2 was evaluated in vitro using molar ratios of inactive Erk-2 to 4-HNE consistent with increasing oxidative stress in vivo. Liquid chromatography combined with tandem mass spectrometry confirmed monomer adduct formation and identified the major adduct species at the histidine 178 residue within the kinase phosphorylation lip. These novel results show that the formation of 4-HNE-Erk-1/2 monomer-adducts results in the inhibition of Erk-Elk-AP-1 signaling in hepatocytes and implicates the His 178 residue with the mechanism of inhibition.

Purpose

To evaluate the added value of non-contrast-enhanced magnetic resonance angiography (MRA) to conventional MR imaging for a detailed characterization of different rodent glioma models.

Materials and Methods

Intracerebral tumor cell implantation and chemical induction methods were implemented to obtain rat C6, 9L/LacZ, F98, RG2 and ENU-induced glioma models, a human U87 MG tumor model as well as a mouse GL261 glioma model. MR assessments were regularly conducted on a 7 Tesla Bruker BioSpin system. The tumor border sharpness and growth characteristics of each glioma model were assessed from T2-weighted images. Neovascularization and vascular alterations inherent to each model were characterized by assessing absolute blood volumes, vessel density, length and diameter using Mathematica and Amira software.

Results

9L/LacZ and ENU gliomas both presented flaws that hinder their use as reliable brain tumor models. C6 gliomas were slightly invasive and induced moderate vascular alterations, whereas GL261 tumors dramatically altered the brain vessels in the glioma region. F98, RG2 and U87 are infiltrative models which produced dramatic vascular alterations.

Conclusion

MRI and MRA provided crucial in vivo information to identify a distinctive “fingerprint” for each of our 7 rodent glioma models.

BACKGROUND: The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. RESULTS: Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. CONCLUSION: This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.