Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.     

Glioma cells under hypoxic conditions block the brain microvascular endothelial cell death induced by serum starvation

Angiogenesis is one of essential components for the growth of neoplasms, including malignant gliomas. However, tumor vascularization is often poorly organized and marginally functional due to tumor structural abnormalities, inducing regional or temporal hypoxic conditions and nutritional shortages in tumor tissues. We investigated how during angiogenesis migrating endothelial cells survive in these hypoxic and reduced nutritional conditions. Human brain microvascular endothelial cells (HBMECs) underwent apoptosis and necrosis after serum withdrawal. This endothelial cell death was blocked by recombinant VEGF protein or the culture medium of U251 glioma cells exposed to hypoxia (H-CM). Hypoxic treatment increased vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-alpha) expression in U251 glioma cells. H-CM activated nuclear factor-kappaB (NFkappaB) protein and increased the gene expression of antiapoptotic factors including Bcl-2, Bcl-X(L), survivin and X-chromosome-linked inhibitor of apoptosis protein (XIAP) in endothelial cells. The survival activity of H-CM for endothelial cells was abolished by two kinds of VEGF inhibitors {Cyclopeptidic VEGF inhibitor and a VEGF receptor tyrosine kinase inhibitor (4-[(4'-chloro-2'-fluoro) phenylamino]-6, 7-dimethoxyquinazoline)} or NFkappaB inhibitors (ALLN and BAY 11-7082). These VEGF inhibitors did not block the activation of NFkappaB induced by H-CM in endothelial cells. On the contrary, TNF-alpha antagonist WP9QY enhanced the survival activity of H-CM for endothelial cells and blocked NFkappaB activation induced by H-CM under serum-starved conditions. Taken together, our data suggest that both the secretion of VEGF from glioma cells and activation of NFkappaB in endothelial cells induced by TNF-alpha are necessary for endothelial cell survival as they increase the expression of antiapoptotic genes in endothelial cells under conditions of serum starvation. These pathways may be one of the mechanisms by which angiogenesis is maintained in glioma tissues.     

Single nucleotide polymorphisms within the promoter region of the rhesus monkey tumor necrosis factor-alpha gene

The human TNF-alpha gene is characterized by several single nucleotide polymorphisms (SNPs) in its promoter region, in part having been shown to influence TNF-alpha expression and susceptibility to various diseases. The rhesus macaque is widely used as an animal model for a variety of TNF-alpha associated pathological conditions, but little is known about genetic variation within the TNF-alpha promoter region. In order to check for such polymorphisms, primers based on rhesus sequence within 5 UTR were designed and used to amplify a 1 kb product from genomic DNA of 29 animals. Sequencing and cloning revealed a total of 11 polymorphisms leading to five different haplotypes.     

TNF/LTα double knockout mice display abnormal inflammatory and regenerative responses to acute and chronic liver injury

Following acute liver injury, hepatocytes divide to facilitate regeneration. However, during chronic injury, hepatocyte proliferation is typically blocked and repair is mediated through liver progenitor (oval) cells. Signalling of the p55 tumour necrosis factor (TNF) receptor is central to these processes. Two ligands for p55 are known: TNF and lymphotoxin-alpha (LT). However, one study suggests that another exists that mediates liver injury following viral challenge. We have therefore investigated whether ligands other than TNF and LT are required for liver regeneration following either acute or chronic injury. Wild-type and double TNF/LT knockout (TNF^–/–LT^–/–) mice were subjected to either partial hepatectomy (PHx) or a choline-deficient ethionine-supplemented (CDE) diet. Proliferating hepatocytes, oval cells and inflammatory cells were identified and quantified in liver sections by immunohistochemistry. Liver inflammatory cells were characterised by cell surface antigen expression. Liver damage and mortality were monitored. Both hepatocyte and oval cell proliferation was reduced in TNF^–/–LT^–/– mice. Lymphocyte clusters were evident in all TNF^–/–LT^–/– livers and were heterogeneous, comprising B and T lymphocytes. PHx evoked liver inflammation in TNF^–/–LT^–/– but not wild-type mice, whereas no difference was apparent between genotypes in CDE experiments. Thus, TNF/LT signalling mediates liver regeneration involving both hepatocytes and progenitor cells. The hyper-inflammatory response following PHx in TNF^–/–LT^–/– animals, which is absent following CDE-induced injury, demonstrates that the two forms of liver injury evoke discrete inflammatory responses and provides a model in which such differences can be examined further.     

Differential role of hydrogen peroxide and staurosporine in induction of cell death in glioblastoma cells lacking DNA-dependent protein kinase

Various DNA double-strand break repair mechanisms, in which DNA-dependent protein kinase (DNA-PK) has a major role, are involved both in the development and treatment of glioblastoma. The aim of the present study was to investigate how glioblastoma cells responded to hydrogen peroxide and staurosporine (STS) and how such a response is related to DNA-PK. Two human glioblastoma cell lines, M059J cells that lack DNA-PK activity, and M059K cells that express a normal level of DNA-PK, were exposed to hydrogen peroxide or STS. The response of the cells to hydrogen peroxide or STS was recorded by measuring cell death, which was detected by three different methods—MTT, annexin-V and propidium iodide staining, and JC-1 mitochondrial probe. The result showed that both hydrogen peroxide and STS were able to induce cell death of the glioblastoma cells and that the former was mainly associated with necrosis and the latter with apoptosis. Glioblastoma cells lacking DNA-PK were less sensitive to STS treatment than those containing DNA-PK. However, DNA-PK had no significant influence on hydrogen peroxide treatment. We further found that catalase, an antioxidant enzyme, could prevent cell death induced by hydrogen peroxide but not by STS, suggesting that the pathways leading to cell death by hydrogen peroxide and STS are different. We conclude that hydrogen peroxide and STS have differential effects on cell death of glioblastoma cells lacking DNA-dependent protein kinase. Such differential roles in the induction of glioblastoma cell death can be of significant value in selecting and/or optimizing the treatment for this malignant brain tumor.     

Germinal vesicle materials are requisite for male pronucleus formation but not for change in the activities of CDK1 and MAP kinase during maturation and fertilization of pig oocytes

In amphibian oocytes, it is known that germinal vesicle (GV) materials are essential for sperm head decondensation but not for activation of MPF (CDK1 and cyclin B). However, in large animals, the role of GV materials in maturation and fertilization is not defined. In this study, we prepared enucleated pig oocytes at the GV stage and cultured them to examine the activation and inactivation of CDK1 and MAP kinase during maturation and after electro-activation. Moreover, enucleated GV-oocytes after maturation culture were inseminated or injected intracytoplasmically with spermatozoa to examine their ability to decondense the sperm chromatin. Enucleated oocytes showed similar activation/inactivation patterns of CDK1 and MAP kinase as sham-operated oocytes during maturation and after electro-stimulation or intracytoplasmic sperm injection. During the time corresponding to MI/MII transition of sham-operated oocytes, enucleated oocytes inactivated CDK1. However, penetrating sperm heads in enucleated oocytes did not decondense enough to form male pronuclei. To determine whether the factor(s) involved in sperm head decondensation remains associated with the chromatin after GV breakdown (GVBD), we did enucleation soon after GVBD (corresponding to pro-metaphase I, pMI) to remove only chromosomes. The injected sperm heads in pMI-enucleated oocytes decondensed and formed the male pronuclei. These results suggest that in pig oocytes, GV materials are not required for activation/inactivation of CDK1 and MAP kinase, but they are essential for male pronucleus formation.     

Oncostatin M induces an acute phase response but does not modulate the growth or maturation-status of liver progenitor (oval) cells in culture

Following acute injury, the liver regenerates through hepatocyte division. If this pathway is impaired, liver repair depends on the recruitment of adult liver progenitor (oval) cells. Mice fed a choline deficient, ethionine supplemented (CDE) diet possess substantial numbers of oval cells, which can be isolated, or examined in vivo. Oncostatin M (OSM) has been shown to induce maturation of murine fetal hepatoblasts into hepatocytes. We recently confirmed this in human fetal liver cultures. Here, we show that liver OSM expression increases in mice fed a CDE diet and CDE-derived oval cell isolates express OSM and its receptor (OSMR). Oval cell lines (PIL cells), as well as primary oval cell cultures, displayed STAT-3 phosphorylation following OSM stimulation. OSM had no effect on the growth of primary oval cells, but it was pro-apoptotic to PIL cells, suggesting that the two cell models are not directly comparable. Expression of PCNA and cyclin D1 was not affected by OSM treatment. No evidence was obtained to suggest an effect on oval cell maturation with OSM treatment. However, decreased albumin production, accompanied by increased expression of haptoglobin and fibrinogen, suggests that OSM induced an acute phase reaction in cultured oval cells.     

NMR structure and regulated expression in APL cell of human SH3BGRL3

SH3 domain binding glutamic acid-rich protein like 3 (SH3BGRL3) is the new member of thioredoxin (TRX) super family, whose posttranslational modified form was identified as tumor necrosis factor alpha (TNF-alpha) inhibitory protein, TIP-B1. In this paper, we determined its solution structure by multi-dimensional nuclear magnetic resonance spectroscopy. The overall structure of human SH3BGRL3 conformed to a TRX-like fold. To understand its function in vivo, the upregulated expression in acute promyelocytic leukemia cell line NB4 at both mRNA and protein level was elucidated. Immunofluorescence and immunohistochemistry staining with monoclonal antibody against SH3BGRL3 demonstrated that it was a cytoplasmic protein in both NB4 cell and human tissues. These results, as a whole, indicate that SH3BGRL3 may function as a regulator in all-trans retinoic acid-induced pathway.     

Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR

This study quantified Neospora caninum DNA in the blood and brain of pregnant and aborted heifers by monitoring PCR product formation in real-time using SYBR Green I, a double-stranded DNA-binding dye. Primers were designed to amplify a 188 bp product specific to N. caninum from the Nc-5 gene fragment of N. caninum. Similarly, a 71 bp product was amplified from the 28S rRNA gene of bovine genomic DNA that served as a control. Agarose gel electrophoresis and analysis of the melting curve for PCR products showed that both primer pairs were specific to their targets. Standard curves were generated for both bovine and N. caninum genomic DNA, and were used to compute the relative concentration of parasite to bovine DNA in the test samples. The concentration of N. caninum DNA in 1 ng of bovine genomic DNA obtained from blood ranged between 0.097 ng at the 1st week of the observation and 0 ng at the 15th week in aborted cows. In pregnant cows, the values ranged between 0.080 ng at the 1st week and 0.155 ng at the 15th week of observation. There was a sustained decrease of DNA concentration in the aborted group after abortion and an increase in DNA concentration in the pregnant group. Comparison of parasite DNA in blood and brain of infected heifers showed a higher DNA concentration in brain than in blood. This study shows that N. caninum DNA can be quantified to obtain the relative concentration of parasite DNA to host genomic DNA in blood. This technique allows testing of a large number of samples at one time and can be done without the need for slaughter of tested animals.