Relationships of Educational Attainment and Household Food Insecurity with Obesity: Findings from the 2007-2016 National Health and Nutrition Examination Survey.

This study aimed to determine whether 1882 Black young adults' educational attainment was associated with their obesity ([BMI] ≥ 30) and whether this association varied with household food insecurity. Data from interviews with Black young adults and a medical examination from the 2007-2016 National Health and Nutrition Examination Survey were analyzed. Modified Poisson regressions with robust standard errors were used. Educational attainment was not associated with obesity (prevalence ratio [PR] = 1.05, 95% confidence interval [CI]: 0.85, 1.30) after adjusting for age, sex, marital status, smoking status, drinking status, income, health insurance status, physical activity level, and household food insecurity. The interaction between educational attainment and household food insecurity was also not significant (PR = 1.11, 95% CI: 0.56, 2.19) after adjusting for the same covariates. These findings indicated that college graduates were as likely to be obese as those with less education, and the relationship between educational attainment and obesity did not vary with household food insecurity. Future studies should conduct longitudinal analyses of these relationships. There is a need to identify the roles that education, household food insecurity, and other measures of socioeconomic status play in Black young adults' obesity.

CBP/catenin antagonist safely eliminates drug-resistant leukemia-initiating cells.

CREB-binding protein (CBP) and p300 are highly homologous transcriptional coactivators with unique, non-redundant roles that bind a wide array of proteins, including catenins-ß and γ. ICG-001 is a small-molecule inhibitor that specifically inhibits the CBP/catenin interaction. Importantly, ICG-001 does not inhibit the p300/catenin interaction. We demonstrate that specifically inhibiting the interaction between CBP and catenin with ICG-001 results in the differentiation of quiescent drug-resistant chronic myelogenous leukemia-initiating cells (CML LICs), thereby sensitizing them to BCR-ABL tyrosine kinase inhibitors, for example, Imatinib. Using ICG-001 in a NOD/SCID/IL2Rγ(-/-) mouse model of engrafted human chronic myelogenous leukemia, we now demonstrate the complete elimination of engrafted leukemia after only one course of combined chemotherapy. Combination-treated animals live as long as their non-engrafted littermates. Results from these studies demonstrate that specifically antagonizing the CBP/catenin interaction with ICG-001 can eliminate drug-resistant CML LICs without deleterious effects to the normal endogenous hematopoietic stem cell population.

A population-based estimate of the substantial burden of diarrhoeal disease in the United States; FoodNet, 1996-2003.

From 1996 to 2003, four 12-month population-based surveys were performed in FoodNet sites to determine the burden of diarrhoeal disease in the population. Acute diarrhoeal illness (ADI) was defined as > or =3 loose stools in 24 hours with impairment of daily activities or duration of diarrhoea >1 day. A total of 52840 interviews were completed. The overall weighted prevalence of ADI in the previous month was 5.1% (95% CI+/-0.3%), corresponding to 0.6 episodes of ADI per person per year. The average monthly prevalence of ADI was similar in each of the four survey cycles (range 4.5-5.2%). Rates of ADI were highest in those age <5 years. Of those with ADI, 33.8% (95% CI+/-2.7%) reported vomiting, 19.5% (95% CI+/-2.1%) visited a medical provider, and 7.8% (95% CI+/-1.4%) took antibiotics. Rates of ADI were remarkably consistent over time, and demonstrate the substantial burden placed on the health-care system.

On methods for gene function scoring as a means of facilitating the interpretation of microarray results.

As gene annotation databases continue to evolve and improve, it has become feasible to incorporate the functional and pathway information about genes, available in these databases into the analysis of gene expression data, for a better understanding of the underlying mechanisms. A few methods have been proposed in the literature to formally convert individual gene results into gene function results. In this paper, we will compare the various methods, propose and examine some new ones, and offer a structured approach to incorporating gene function or pathway information into the analysis of expression data. We study the performance of the various methods and also compare them on real data, using a case study from the toxicogenomics area. Our results show that the approaches based on gene function scores yield a different, and functionally more interpretable, array of genes than methods that rely solely on individual gene scores. They also suggest that functional class scoring methods appear to perform better and more consistently than overrepresentation analysis and distributional score methods.

An improved resazurin-based cytotoxicity assay for hepatic cells.

A simple resazurin-based cytotoxicity assay is presented for screening of cytotoxicity in hepatocytes and liver cell lines. Human hepatoma (HepG2) cells in 96-well culture plates were exposed to known toxic (cisplatin, 5-fluorouracil, ethionine, flufenamic acid, and diflunisal) and control (transplatin, 5-chlorouracil, methionine, and acetylsalicylic acid) compounds for 1-3 days, and resazurin (5 micromol/L) was added. A conventional short-term (1 h) assay was first performed, where cytotoxicity is indicated by decreased reduction of resazurin to its fluorescent product resorufin. Our improved assay consists of additionally measuring fluorescence 2-4 days later, when cytotoxicity is indicated by a striking increase in the concentration of resorufin, resulting from two distinct processes. First, viable liver-derived cells slowly convert resorufin to nonfluorescent metabolites. Fluorescence of control cell wells decreased to background during a 2- to 4-day exposure to resazurin. This metabolism of resorufin was largely blocked by dicumarol and to lesser extents by disulfiram and SKF525a. Second, dead or dying cells slowly convert resazurin to resorufin but do not further metabolize resorufin; thus this fluorescent metabolite accumulates to high levels in wells with dead cells by 2 to 4 days. A similar increase in fluorescence associated with cytotoxicity was observed in primary cultures of rat hepatocytes using the long-term resazurin-based assay. In addition to an improved signal relative to the short-term assay, the inversion of the fluorescent signal from high = alive short-term to high = dead long-term allows determination of two independent cytotoxicity endpoints after addition of one innocuous vital dye.

Apolipoprotein E and mimetic peptide initiate a calcium-dependent signaling response in macrophages.

Apolipoprotein E (ApoE) is a 34-kDa cholesterol transport protein that also possesses immunomodulatory properties. In this study, we demonstrate that ApoE initiates a signaling cascade in murine peritoneal macrophages that leads to increased production of inositol triphosphate with mobilization of intracellular Ca(2+) stores. This cascade is inhibited by pretreatment with receptor-associated protein and Ni(2+), and it is mediated by a pertussis toxin-sensitive G protein. These properties are characteristic of signal transduction induced via ligand binding to the cellular receptor, lipoprotein receptor-related protein. A peptide derived from the receptor-binding region of ApoE also initiates signal transduction in a manner similar to that of the intact protein, suggesting that this isolated region is sufficient for signal transduction. The ApoE-mimetic peptide competed for binding with the intact protein, confirming that they both interact with the same site. ApoE-dependent signal transduction might play a role in mediating the functional properties of this lipoprotein.

Cytochrome P450 induction in rat hepatocytes assessed by quantitative real-time reverse-transcription polymerase chain reaction and the RNA invasive cleavage assay.

The acceleration of drug discovery due to combinatorial chemistry and high-throughput screening methods has increased the numbers of candidate pharmaceuticals entering the drug development phase, and the capability to accurately predict whether drug candidates will induce various members of the drug-metabolizing cytochrome P450 (CYP) enzyme superfamily is currently of great interest to the pharmaceutical industry. In the present study, we describe the rapid and reliable analysis of CYP induction in a readily obtained model system (cultured rat hepatocytes) using both real-time quantitative reverse transcription-polymerase chain reaction (real-time RT-PCR) and the RNA invasive cleavage assay. The levels of members in the three primary inducible rat CYP subfamilies (CYP1A1, CYP2B1/2, and CYP3A1) were analyzed in untreated and induced (beta-naphthoflavone, phenobarbital, and hydrocortisone) hepatocyte cultures under various media conditions to screen for optimal CYP induction profiles. The fold inductions measured by real-time RT-PCR and the RNA invasive cleavage assay were also compared with enzyme activity measurements in parallel cultures using liquid chromatography/double mass spectrometry-based assays, and the sensitivity and the specificity of the two RNA analysis methods were compared. Using these techniques, various culture conditions were examined for optimizing induction of the three CYP subfamily members. Both real-time RT-PCR and the RNA invasive cleavage assay prove to be effective methods for determining the effects of drugs on specific CYPs in primary rat hepatocytes.

Protection against glutamate toxicity through inhibition of the p44/42 mitogen-activated protein kinase pathway in neuronally differentiated P19 cells.

Excessive levels of the neurotransmitter glutamate trigger excitotoxic processes in neurons that lead to cell death. N-Methyl-D-aspartate (NMDA) receptor over-activation is a key excitotoxic stimulus that leads to increases in intracellular calcium and activation of downstream signaling pathways, including the p44/42 mitogen-activated protein (MAP) kinase pathway. In the present study, we have demonstrated that 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a potent and selective inhibitor of the p44/42 MAP kinase signaling pathway, prevents glutamate-induced death in neuronally differentiated P19 cells. In addition, we show that differentiated, but not undifferentiated, P19 cells expressed zeta1, epsilon1, and epsilon2 subunits of the NMDA receptor. Differentiated P19 cells exhibited specific NMDA receptor binding and intracellular calcium responses to glutamate that were blocked by the selective NMDA receptor antagonist [5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801), but not U0126. Glutamate treatment of differentiated P19 cells triggered a rapid and sustained induction in p42 MAP kinase phosphorylation that was blocked by U0126. Pretreatment of differentiated P19 cells with U0126, but not other classes of protein kinase inhibitors, protected against glutamate-induced cell death. Post-treatment with U0126, even as late as 6 hr after glutamate application, also protected against glutamate toxicity. These results suggest that the p44/42 MAP kinase pathway may be a critical downstream signaling pathway in glutamate receptor-activated toxicity.

Nile Red binding to HepG2 cells: an improved assay for in vitro studies of hepatosteatosis.

Nile Red is a fluorescent dye used extensively to study fat accumulation in many types of cells; unfortunately protocols that work well for most cells are not effective for studying drug-induced lipid accumulation in cultured liver cells and hepatocyte-derived cell lines. Using human hepatoma (HepG2) cells, we have developed a simple Nile Red binding assay as a screen for steatosis-inducing compounds. Increases in Nile Red binding in response to known hepatotoxic compounds were observed after incubating treated cells with 1 microM Nile Red for several hours, washing away free Nile Red, and then allowing redistribution, and/or clearance of the lipid-indicator dye. Several compounds known to cause hepatic fat accumulation in vivo were examined and most robustly increased Nile Red binding in HepG2 cells. These include estrogen and other steroids, ethionine, cyclosporin A, and valproic acid. Required concentrations for increased Nile Red binding were generally three-fold or more lower than the cytotoxic concentration determined by a resazurin reduction assay in the same cells. Qualitatively similar Nile Red binding results were obtained when primary canine or rat hepatocytes were used. Morphological differences in Nile Red staining were observed by confocal fluorescence microscopy in HepG2 cells after treatment with different compounds and likely reflect distinct toxicological mechanisms.

Toxicogenomics-based discrimination of toxic mechanism in HepG2 human hepatoma cells.

The rapid discovery of sequence information from the Human Genome Project has exponentially increased the amount of data that can be retrieved from biomedical experiments. Gene expression profiling, through the use of microarray technology, is rapidly contributing to an improved understanding of global, coordinated cellular events in a variety of paradigms. In the field of toxicology, the potential application of toxicogenomics to indicate the toxicity of unknown compounds has been suggested but remains largely unsubstantiated to date. A major supposition of toxicogenomics is that global changes in the expression of individual mRNAs (i.e., the transcriptional responses of cells to toxicants) will be sufficiently distinct, robust, and reproducible to allow discrimination of toxicants from different classes. Definitive demonstration is still lacking for such specific "genetic fingerprints," as opposed to nonspecific general stress responses that may be indistinguishable between compounds and therefore not suitable as probes of toxic mechanisms. The present studies demonstrate a general application of toxicogenomics that distinguishes two mechanistically unrelated classes of toxicants (cytotoxic anti-inflammatory drugs and DNA-damaging agents) based solely upon a cluster-type analysis of genes differentially induced or repressed in cultured cells during exposure to these compounds. Initial comparisons of the expression patterns for 100 toxic compounds, using all approximately 250 genes on a DNA microarray ( approximately 2.5 million data points), failed to discriminate between toxicant classes. A major obstacle encountered in these studies was the lack of reproducible gene responses, presumably due to biological variability and technological limitations. Thus multiple replicate observations for the prototypical DNA damaging agent, cisplatin, and the non-steroidal anti-inflammatory drugs (NSAIDs) diflunisal and flufenamic acid were made, and a subset of genes yielding reproducible inductions/repressions was selected for comparison. Many of the "fingerprint genes" identified in these studies were consistent with previous observations reported in the literature (e. g., the well-characterized induction by cisplatin of p53-regulated transcripts such as p21(waf1/cip1) and PCNA [proliferating cell nuclear antigen]). These gene subsets not only discriminated among the three compounds in the learning set but also showed predictive value for the rest of the database ( approximately 100 compounds of various toxic mechanisms). Further refinement of the clustering strategy, using a computer-based optimization algorithm, yielded even better results and demonstrated that genes that ultimately best discriminated between DNA damage and NSAIDs were involved in such diverse processes as DNA repair, xenobiotic metabolism, transcriptional activation, structural maintenance, cell cycle control, signal transduction, and apoptosis. The determination of genes whose responses appropriately group and dissociate anti-inflammatory versus DNA-damaging agents provides an initial paradigm upon which to build for future, higher throughput-based identification of toxic compounds using gene expression patterns alone.

Endogenous apolipoprotein E suppresses LPS-stimulated microglial nitric oxide production.

The human apolipoprotein (apo) E4 isoform is associated with an increased risk for Alzheimer's disease (AD) and poor prognosis after acute CNS injury. Addition of human apoE inhibits murine microglial activation in culture, suggesting that microglia might be an important physiological target of apoE. In the present study, we examined the role of endogenous murine apoE in modulating microglial nitric oxide (NO) production following lipopolysaccharide (LPS) stimulation. Brain cultures from apoE-deficient mouse pups showed enhanced NO production relative to cultures from wild-type mice and from transgenic mice expressing the human apoE3 isoform, demonstrating that endogenous apoE produced by glial cultures is capable of inhibiting microglial function. ApoE produced within the brain may suppress microglial reactivity and thus alter the CNS response to acute and chronic injury.

Role of protein kinase C in microglia-induced neurotoxicity in mesencephalic cultures.

Microglial activation selectively kills certain neuron populations in mixed neuronal/glial cultures, which may prove useful for modeling neurodegenerative diseases such as Parkinson's disease. In mesencephalic mixed neuronal/glial cultures, microglial activation by zymosan A killed more dopaminergic neurons, assessed by [3H]dopamine uptake and by counting tyrosine hydroxylase-immunoreactive neuron number, than did microglial activation by lipopolysaccharide (LPS). The additional toxicity of zymosan resulted from microglial protein kinase C (PKC) activation. Both zymosan and PMA, but not LPS, activated PKC in enriched microglial preparations. In the mixed neuronal/glial cultures, activation of PKC by phorbol myristate acetate (PMA) increased LPS-induced nitric oxide (NO; by nitrite measurements), but not zymosan-induced NO production, and increased LPS-induced dopaminergic neurotoxicity, but not zymosan-induced dopaminergic neurotoxicity. Additive effects of PMA and LPS, similar to zymosan effects alone, reflected activation of distinct neurotoxic pathways in the microglia. The NO synthase inhibitor N-nitro-L-arginine methyl ester (NAME) totally blocked the neurotoxicity of LPS, and partially blocked zymosan-induced neurotoxicity; NAME did not block the PKC component of neurotoxicity. In addition to stimulating NO production as effectively as LPS, zymosan also activates microglial PKC and associated non-NO-mediated neurotoxic pathways that may be important in human neurodegenerative diseases. Since the role of NO in human microglia-induced neurotoxicity is controversial, zymosan may prove more useful than LPS as a microglial activator in the rodent mixed neuronal/glial culture model.

Characterization of a CNS cell line, CAD, in which morphological differentiation is initiated by serum deprivation.

A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.

Inhibition of lipopolysaccharide-induced nitric oxide and cytokine production by ultralow concentrations of dynorphins in mixed glia cultures.

Dynorphins (dyn) are a major class of endogenous opioid peptides that modulate the functions of immune cells. However, the effects of dyn on the immune functions of glial cells in the central nervous system (CNS) have not been well characterized. Because nitric oxide (NO) and the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) produced by glial cells are involved in various physiopathological conditions in the CNS, this study examined the effects of dyn on the production of NO and TNF-alpha from mouse glial cells treated with lipopolysaccharide (LPS). LPS induced a concentration-dependent increase in the production of NO or TNF-alpha from the mouse primary mixed glia cultures. Ultralow concentrations (10(-16)-10(-12) M) of dynorphin (dyn) A-(1-8) significantly inhibited the LPS-induced production of NO or TNF-alpha. The inhibitory effects of dyn A-(1-8) were not blocked by nor-binaltorphimine, a selective kappa opioid receptor antagonist. U50-488H, a selective kappa opioid receptor agonist, did not affect the LPS-induced production of NO or TNF-alpha. Ultralow concentrations (10(-16)-10(-12) M) of des-[Tyr1]-dyn A-(2-17), a nonopioid analog that does not bind to kappa opioid receptors, exhibited the same inhibitory effects as dyn A-(1-17) and dyn A-(1-8). These results suggest that dyn modulate the immune functions of microglia and/or astrocytes in the brain and these modulatory effects of dyn are not mediated by classical kappa opioid receptors.

The effects of the HIV-1 envelope protein gp120 on the production of nitric oxide and proinflammatory cytokines in mixed glial cell cultures.

Although the neurotoxicity induced by the HIV envelope protein, gp120, has been demonstrated to require the presence of glial cells (microglia/astrocytes), the mechanisms for the gp120-induced neurotoxicity are not well understood. Moreover, the neurotoxic potencies of gp120s obtained from various HIV isolates are different. Since nitric oxide (NO) and proinflammatory cytokines (TNF-alpha, IL-1, IL-6) produced by glial cells have been involved in the neuropathogenesis of various diseases, this study examined the effects of gp120 obtained from two strains, HIV-1IIIB and HIV-1SF2, of the HIV-1 virus on the production of NO, TNF-alpha, IL-1 alpha, IL-1 beta, and IL-6 in murine primary mixed glial cell cultures. The glial cells exposed to HIV-1IIIB gp120 released NO, TNF-alpha, and IL-6 in a dose-dependent manner, whereas IL-1 alpha and IL-1 beta were undetectable. The cells exposed to HIV-1SF2 gp120 increased the release of IL-6 only. The gp120-induced effects were significantly enhanced by priming glial cells with IFN-gamma. To investigate the cellular sources and mechanisms of the gp120-induced IL-6 production, in situ hybridization with mRNA for IL-6 was performed in HIV-1IIIB gp120- or HIV-1SF2 gp120-stimulated microgliaenriched or astrocyte-enriched cultures. HIV-1IIIB gp120 or HIV-1SF2 gp120 induced the expression of IL-6 mRNA in both microglia-enriched and astrocyte-enriched cultures, indicating that both microglia and astrocytes produce IL-6, and that the transcriptional regulation is involved in the gp120-induced IL-6 production. Taken together, these results demonstrate that the production of NO, TNF-alpha, IL-1, or IL-6 from glial cells is differentially regulated by HIV-1IIIB gp120 and HIV-1SF2 gp120. These results may provide insights into the roles of NO and proinflammatory cytokines in the neurotoxicity of gp120s and the neuropathology of different strains of HIV-1 viruses.

Long-term expression of the 35,000 mol. wt fos-related antigen in rat brain after kainic acid treatment.

Systemic injection of kainic acid, a rigid analogue of glutamate, induces both the short-term and the long-term expression of activator protein-1 transcription factors. The short-term responses of activator protein-1 factors such as c-fos and fos-related antigens have been well studied. However, the long-term expression of activator protein-1 factor(s) induced by kainic acid is poorly understood. The present study was designed to document the long-term expression (up to seven months) of the fos-related antigens and to map their distributions in the rat brain after systemic treatment with kainic acid. A single dose of kainic acid (8 mg/kg) was injected i.p. into Fischer 344 rats and their epileptic seizure behaviour was monitored. The rats with full limbic seizures were chosen for long-term study. By using immunocytochemistry with an antibody that cross-reacts with all known fos-related antigens, western blot analysis and a gel mobility-shift assay, we have now shown that a 35,000 mol. wt fos-related antigen was induced by kainic acid treatment and expressed at high levels for up to five months. This fos-related antigen still maintains the activator protein-1 DNA binding activity in the rat brain seven months after kainic acid treatment. The fos-related antigens and activator protein-1 binding activity were continuously expressed at high levels throughout the experimental period in the dentate granule cells where mossy fibre collateral sprouting occurred after kainic acid treatment. Our results suggested that long-term expression of fos-related antigen may reflect the pathophysiological changes after kainic acid administration.

Dexamethasone and forskolin synergistically increase [Met5]enkephalin accumulation in mixed brain cell cultures.

Possible synergistic effects of the glucocorticoid dexamethasone (DEX, 10(-7) M) and the adenylate cyclase agonist forskolin (FSK, 10(-5) M) on [Met5]enkephalin (ME) accumulation were examined in enriched rat glial cultures and in mixed neuronal/glial cultures. In enriched glial cultures, DEX and FSK each stimulated the accumulation of ME 2-3-fold over basal media levels, but there was little additional stimulation when these agonists were combined. In contrast, mixed neuronal/glial cultures showed only weak responses to DEX or FSK alone, but the combination of these agonists produced a pronounced synergistic effect on media ME accumulation (6-10-fold over basal levels). The DEX effect was mediated via a classical glucocorticoid receptor, since DEX was potent (acting over a concentration range of 10(-11)-10(-7) M), mimicked by corticosterone (10(-6) M), and blocked by the glucocorticoid receptor antagonist RU486. There was a pronounced time lag (2 days) for the synergistic effects of DEX + FSK to develop. In situ hybridization and immunocytochemical studies suggested that astrocytes were the major source for the increased ME production in all mixed neuronal/glial cultures examined. Creating a mixed culture by plating fetal neurons onto confluent, enriched P7 glial cultures inhibited accumulation of ME in the media. DEX + FSK, but neither agonist alone, overcame this neuronal inhibition and increased accumulation of media ME to levels identical to levels in stimulated enriched glial cultures. The net effect was a 6-fold increase in ME accumulation in the mixed neuronal/glial cultures relative to a 2.5-fold increase in the enriched glial cultures. Neuronal inhibition of basal glial ME production could explain the similar synergistic effects of DEX + FSK observed in all mixed neuronal/glial cultures examined, and may be important in suppressing ME production by astrocytes in the brain.

Protein tyrosine kinase inhibitors suppress the production of nitric oxide in mixed glia, microglia-enriched or astrocyte-enriched cultures.

Nitric oxide (NO) produced by glial cells has been implicated in the neuropathogenesis of various diseases. However, the signaling transduction pathway(s) for the production of NO in these cells is not well understood. To test whether protein tyrosine kinases (PTKs) are required for signaling events of NO production in glial cells, this study examined the effects of genistein and tyrphostin A25, two potent inhibitors of PTKs, on the production of NO in mouse primary mixed glia, microglia-enriched or astrocyte-enriched cultures exposed to lipopolysaccharide (LPS) or a combination of LPS and interferon-gamma (IFN gamma). LPS induced a dose-dependent increase in NO production from the mixed glia cultures. The LPS-induced NO production was significantly enhanced by stimulating the cells with IFN gamma. Genistein or tyrphostin A25 inhibited the production of NO in both LPS- and IFN gamma/LPS-stimulated mixed glia cultures. The production of NO in the stimulated microglia-enriched or astrocyte-enriched cultures was also inhibited by tyrphostin A25. To verify the cellular sources of NO, immunocytochemical staining of inducible NO synthase (iNOS) was followed by staining with the microglia marker Mac-1 or the astrocyte marker glial fibrillary acid protein (GFAP) in microglia-enriched or astrocyte-enriched cultures. The expression of iNOS and the production of NO in microglia-enriched cultures were significantly higher than those in the identically stimulated astrocyte-enriched cultures. These results demonstrate that PTKs are involved in the signaling events of LPS-induced NO production in microglia and astrocytes, and that microglia are more responsive than astrocytes to stimuli which induce NO. These results may provide insights into therapeutic interventions in the pathway for NO production in the brain.

Transcription factors in primary glial cultures: changes with neuronal interactions.

Several astrocyte gene products, such as enkephalin and glial fibrillary acidic protein (GFAP), are expressed at higher levels under in vitro conditions relative to in vivo. We have observed that cultured glial cells express high basal levels of transcription factors, such as fos-related antigens (Fra), c-Jun, JunD, and cAMP responsive element binding protein (CREB). When neuronal cells are plated on top of the monolayers, the expression of Fra, c-Jun, JunD, and GFAP decreases in the astroglial cells. The DNA binding activity to the AP-1-like sites of the GFAP and proenkephalin genes was examined in these cultures. The protein complex from glial cultures which recognizes the GFAP AP-1 element contained Fra immunoreactivity while the DNA binding from mixed neuronal/glial cultures consists of CREB-immunoreactive proteins. In glial cultures, no binding occurred to the proenkephalin AP-1-like element but a CREB-immunoreactive complex recognized this sequence in the mixed cultures. Thus, with the addition of neurons, both transcription factors and target gene products decrease in astroglial cells. The proteins that compose gene modulatory complexes also change suggesting that regulation of astroglial gene expression is modulated by neurons.

Induction of NF-kB-like transcription factors in brain areas susceptible to kainate toxicity.

Administration of kainate (KA), a glutamate receptor agonist, to rats causes neuronal damage in the CA1/CA3 fields of the hippocampus and in the pyriform/ entorhinal cortex. Reactive gliosis also occurs and activated astrocytes upregulate their expression of a large number of molecules. Since NF-kB transcription factors are involved in cellular responses to diverse pathogenic stimuli and have been shown to be induced in astrocytes in vitro in response to cytokines and growth factors, we investigated their possible involvement in the changes in gene expression subsequent to KA-induced lesions. Immunoreactivity to the p65 subunit of NF-kB was markedly increased in non-neuronal cells 2 days after KA administration (8 mg/kg i.p.) in the areas of selective neuronal degeneration. This increase was not observed 3 h or 1 day after injection, but was still present 7-10 days after KA injection. By gel mobility-shift assay, a protein complex binding to the kB consensus sequence was found to be induced by 2 days after KA, which correlated with immunohistochemical findings. This NF-kB-protein complex seemed to be localized in reactive astrocytes, as indicated by the morphological similarity of NF-kB-positive cells and reactive astrocytes stained with glial fibrillary acidic protein (GFAP) antibody, and the parallelism between the time course of NF-kB induction and appearance of gliosis after KA treatment. Double immunocytochemistry experiments demonstrated the colocalization of NF-kB positive cells and reactive astrocytes. Our results suggest that activated NF-kB in astrocytes participates in delayed and long-term responses of glia to injury.