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Direct contact and communication between pericytes and endothelial cells is critical for maintenance of cerebrovascular stability and blood-brain barrier function. Capillary pericytes have thin processes that reach hundreds of micrometers along the capillary bed. The processes of adjacent pericytes come in close proximity but do not overlap, yielding a cellular chain with discrete territories occupied by individual pericytes. Little is known about whether this pericyte chain is structurally dynamic in the adult brain. Using in vivotwo-photon imaging in adult mouse cortex, we show that while pericyte somata were immobile, the tips of their processes underwent extensions and/or retractions over days. The selective ablation of single pericytes provoked exuberant extension of processes from neighboring pericytes to contact uncovered regions of the endothelium. Uncovered capillary regions had normal barrier function but were dilated until pericyte contact was regained. Pericyte structural plasticity may be critical for cerebrovascular health and warrants detailed investigation.

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated “donor” cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.

The aim of our study was to compare the angiographic changes in 53 nondiabetic patients, 54 type 2 diabetic patients of less than 5 years of duration, 41 patients with 5–10 years of diabetes, and 27 with more than 10 years of diabetic duration.

Citrus limonoids have shown to inhibit the growth of cancer in colon, lung, mouth, stomach and breast in animal and cell culture studies. For the first time in the present study, an attempt has been made to isolate antioxidant fractions and five limonoids from red Mexican grapefruit seeds. Defatted seed powder was successively extracted with hexane, ethyl acetate (EtOAc), acetone, methanol (MeOH) and MeOH/water and the extracts were concentrated under vacuum. Radical scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and total phenolic content were also measured for comparison with the antioxidant capacity in the phosphomolybdenum method for the above extracts. Acetone and MeOH extracts, respectively, showed the highest (85.7%) and lowest (53.3%) radical scavenging activity, at 500 ppm. The total phenolic contents were found to be highest in the acetone extract (15.94%) followed by the MeOH extract (5.92%), ethyl acetate extract (5.54%) and water extract (5.26%). Antioxidant capacity of the extracts as equivalents to ascorbic acid (µmol/g of the extract) was in the order, EtOAc extract > acetone extract > water extract > methanol extract. Furthermore, the EtOAC and acetone extracts were loaded onto silica gel columns to obtain four limonoid aglycons. MeOH fraction was loaded onto a dowex-50 and sepabeads resin column to obtain a limonoid glucoside. The purity of the isolated five compounds was analyzed by HPLC using a C18 column and UV detection at 210 nm. Finally, the structures of the compounds were identified as obacunone, nomilin, limonin, deacetylnomilin (DAN) and limonin-17-ß-d-glucopyranoside (LG) using 1H and 13C NMR studies.

2-Hydroxy fatty acids are relatively minor species of membrane lipids found almost exclusively as N-acyl chains of sphingolipids. In mammals, 2-hydroxy sphingolipids are uniquely abundant in myelin galactosylceramide and sulfatide. Despite the well-documented abundance of 2-hydroxy galactolipids in the nervous system, the enzymatic process of the 2-hydroxylation is not fully understood. To fill this gap, we have identified a human fatty acid 2-hydroxylase gene (FA2H) that is highly expressed in brain. In this report, we test the hypothesis that FA2H is the major fatty acid 2-hydroxylase in mouse brain and that free 2-hydroxy fatty acids are formed as precursors of myelin 2-hydroxy galactolipids. The fatty acid compositions of galactolipids in neonatal mouse brain gradually changed during the course of myelination. The relative ratio of 2-hydroxy versus nonhydroxy galactolipids was very low at 2 days of age (∼8% of total galactolipids) and increased 6- to 8-fold by 30 days of age. During this period, free 2-hydroxy fatty acid levels in mouse brain increased 5- to 9-fold, and their composition was reflected in the fatty acids in galactolipids, consistent with a precursor-product relationship. The changes in free 2-hydroxy fatty acid levels coincided with fatty acid 2-hydroxylase activity and with the upregulation of FA2H expression. Furthermore, mouse brain fatty acid 2-hydroxylase activity was inhibited by anti-FA2H antibodies. Together, these data provide evidence that FA2H is the major fatty acid 2-hydroxylase in brain and that 2-hydroxylation of free fatty acids is the first step in the synthesis of 2-hydroxy galactolipids.

Hypoxia/ischemic brain injury accompanies an inflammatory response involving an activation of glial cells. This study, using an in vitro model, investigated the signaling mechanisms mediating hypoxic responses of the two glial cell types (astrocytes and microglia) in relation to the expression of inducible nitric oxide synthase (iNOS). In cultures of rat brain microglia and astrocytes, hypoxia (8 h) followed by reoxygenation (24 h) (H/O) had little (microglia) or no (astrocytes) effect on the expression of iNOS. However, H/O elicited opposite effects on lipopolysaccharide (LPS) induction of iNOS in the two cell types: it potentiated LPS induction of iNOS in microglia but inhibited this response in astrocytes. Similar differential effects of hypoxia were observed on the production of tumor necrosis factor-α (TNFα). In contrast, there was an upregulation of hemoxygenase- 1 (HO-1), a counter-regulatory pathway, with astrocytes showing a bigger induction than microglia. While hypoxic activation of mitogen-activated protein kinases (MAPKs) was similar in the two glial types, the activation pattern of NFκB was clearly different: hypoxia stimulated the activation of NFκB pathway and NFκB-dependent transcription in microglia but not in astrocytes. Lastly, the two cell types displayed differential vulnerabilities to hypoxia-induced cell death, the astrocytes being relatively more resistant than microglia.

The mitogen-activated protein kinases (MAPKs) play a central role in mediating the activation and transcriptional responses of diverse cells, including glia. c-Jun N-terminal kinase (JNK), a member of the MAPK family, is activated by a variety of stress and proinflammatory signals and in turn phosphorylates its downstream substrates including nuclear factors, leading to transcriptional activation of target genes. There are at least three subtypes of JNK (i.e., JNKs 1–3) that may play isoform-specific roles. This study examined the role of JNK isoforms in the induction of inducible nitric oxide synthase (iNOS) in astrocytes in response to lipopolysachharide (LPS) and interferon (IFN)-γ. While an inhibitor of the JNK pathway (SP600125) inhibited iNOS expression, ectopic expression of a constitutively active form of MEKK1 (MAPK/ERK kinase kinase- 1), an upstream activator of JNK, led to an induction of co-transfected iNOS promoter activity and, in the presence of LPS, to an enhanced expression of iNOS. RNA knockdown studies with JNK subtype-specific short-interfering RNA (siRNA), indicated that JNK1- but not JNK2- nor JNK3-specific siRNA, interfered with LPS/IFNγ induction of iNOS. It is concluded that, of the three JNK forms, JNK1 is the major mediator of iNOS induction and perhaps, inflammatory signaling in general, in glial cells.

Our previous studies have identified mechanisms by which cytokine production, blocked by Ly49G2 receptor cross-linking, can be overridden. In this study we analyzed the regulation of other ITAM-positive receptor signaling on NK, NKT, and T cells and characterized the biochemical pathways involved in this signaling. Our studies demonstrate that cross-linking of NKG2D and NK1.1 results in a synergistic NK IFN-γ response when combined with IL-12 or IL-18. Examination of NKT- and T-cell responses demonstrated that cross-linking of NKG2D and CD3 resulted in potent synergy when combined with IL-12 and, to a lesser degree, with IL-18. We have now found that both the p38 MAP kinase and the ERK-dependent signal transduction pathways are required for the synergistic response. Further mechanistic examination of the synergy indicated a potent up-regulation of total IFN-γ mRNA in the nuclear and the cytoplasmic compartment, but mRNA half-life was not affected. Fifteen minutes of IL-12 pretreatment was sufficient to result in maximal synergistic activation, indicating that the response of the cells to the IL-12 signal was rapid and immediate. Thus, our data demonstrate that multiple convergent signals maximize the innate immune response by triggering complementary biochemical signaling pathways.

Alzheimer's disease (AD) is a progressive senile dementia characterized by deposition of a 4 kDa peptide of 39-42 residues known as amyloid beta-peptide (Aβ) in the form of senile plaques and the microtubule associated protein tau as paired helical filaments. Genetic studies have identified mutations in the Aβ precursor protein (APP) as the key triggers for the pathogenesis of AD. Other genes such as presenilins 1 and 2 (PS1 / 2) and apolipoprotein E (APOE) also play a critical role in increased Aβ deposition. Several biochemical and molecular studies using transfected cultured cells and transgenic animals point to mechanisms by which Aβ is generated and aggregated to trigger the neurodegeneration that may cause AD. Three important enzymes collectively known as 'secretases' participate in APP processing leading to the generation of either Aβ or non-amyloid proteins. However, the mechanisms of neurotoxicity of Aβ and the role of APP function in AD remain important unanswered questions. Although early studies recognized the loss of cholesterol and other lipids in the brain, these findings have been poorly connected with AD pathogenesis, despite the identification of the ε4 allele of APOE as a major risk factor in AD. The recent finding that cholesterol can modulate the yield of potentially toxic Aβ has boosted research on its role in AD. Consequently, several cholesterol-reducing drugs are currently being evaluated for the treatment of AD. The present review summarizes our current understanding of the relationship of AD pathogenesis with cholesterol, lipids and other genetic and environmental risk factors.

PCR-based fluorescent detection assays for the relative and quantitative measurement of gene expression, such as Taq Man™, LUX™, and SYBR Green™, are currently in wide spread use due to their general applicability, low cost, reproducibility, accuracy, and ease of use. One current limitation of quantitative PCR (Q-PCR) is the lack of a fully integrated and high-throughput method for general genomic and diagnostic applications. Here we report a reliable and high-throughput system for the automated extraction of RNA, first-strand cDNA synthesis, quality control measures, consecutive real-time PCR amplification, and primary data analysis. As described, this procedure utilizes commonly available reagents and pre-packaged “kits” for RNA extraction, first strand cDNA synthesis, Q-PCR, liquid handling, and capillary electrophoresis that are generally applicable to a wide variety of robotic platforms. (JALA 2004;9:128-34)

Previous studies have shown that mitogen-activated protein kinase (MAPK) cascades signal the induction of inducible nitric-oxide synthase (iNOS) in glial cells (Bhat, N. R., Zhang, P., Lee, J. C., and Hogan E. L. (1998)J. Neurosci.18, 1633–1641; Bhat, N. R., Zhang, P., and Bhat, A. N. (1999) J. Neurochem.72, 472–478). This study further investigates the role of p38 MAPK in the transcriptional activation of the iNOS gene by transient transfection with constitutively active upstream kinases in the pathway (i.e.MAPK kinase 3 (MKK3b(E)) and MAPK kinase 6 (MKK6b(E)). Expression in C-6 glial cells of either MKK3b(E) or MKK6b(E) resulted in an induction of the activity of a cotransfected rat iNOS promoter-reporter (iNOS-luciferase (Luc)) gene and an enhancement of cytokine-induced expression of iNOS mRNA, both of which were inhibitable by the p38 MAPK inhibitor SB203580. The MKK constructs also induced cAMP response element-mediated (CRE-Luc) and nuclear factor κB-dependent (nuclear factor κB-Luc) transcriptional activities. Transfection with dominant negative (dn) forms of CRE-binding protein (CREB) and CCAAT/enhancer-binding protein (C/EBP), the two CRE-binding transcription factors targeted by the p38 MAPK pathway, resulted in opposite effects; dnCREB enhanced and dnC/EBP inhibited iNOS-Luc parallel to their effects on CRE-Luc. In addition, the induction, by MKK3b(E) and MKK6b(E), of iNOS promoter activity was enhanced by a wild-type activating transcription factor (ATF-2), whereas a phosphorylation-defective form of ATF-2 had a suppressive effect. The results of these molecular studies provide evidence for an important role for the p38 MAPK pathway in the transcriptional activation of the iNOS gene in rat glial cells involving the transcription factors nuclear factor κB, C/EBP, and ATF-2.

We demonstrate that dexamethasone-mediated transcription activation of the cytochrome P-450c27 promoter involves a physical interaction and functional synergy between glucocorticoid receptor (GR) and Ets2 factor. Ets2 protein binding to a “weak” Ets-like site of the promoter is dependent on GR bound to the adjacent cryptic glucocorticoid response element. Coimmunoprecipitation and chemical cross-linking experiments show physical interaction between GR and Ets2 proteins. Mutational analyses show synergistic effects of Ets2 and GR in dexamethasone-mediated activation of the cytochrome P-450c27 promoter. The DNA-binding domain of GR, lacking the transcription activation and ligand-binding domains, was fully active in synergistic activation of the promoter with intact Ets2. The DNA-binding domain of Ets2 lacking the transcription activation domain showed a dominant negative effect on the transcription activity. Finally, a fusion protein consisting of the GR DNA-binding domain and the transcription activation domain of Ets2 fully supported the transcription activity, suggesting a novel synergy between the two proteins, which does not require the transactivation domain of GR. Our results also provide new insights on the role of putative weak consensus Ets sites in transcription activation, possibly through synergistic interaction with other gene-specific transcription activators.

ACTIVATED microglia regulate immune and inflammatory responses in the CNS under a variety of stresses due to infection, injury and disease. In this study, we show that a stress-inducible small heat shock protein, α-crystallin, induces in vitroactivation of microglia cultured from newborn rat brain. Exposure of microglia to α-crystallin resulted in an increased production of nitric oxide (NO) and the expression of the inducible NO synthase (iNOS) as determined by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analyses. α-Crystallin also stimulated the synthesis of the pro-inflammatory cytokine, TNFα. The results presented showing microglial induction of the two key immune regulatory and inflammatory molecules, i.e., NO and TNFα, in response to a stress-inducible protein, suggest a link between environmental stress and the CNS immune response.

Antibodies designed to recognize a 74 amino acid sequence of the N- or C-terminal domain of the rat CB1 cannabinoid receptor detected a 58 kDa protein in immunoblots of brain and various cells known to express the CB1 cannabinoid receptor. A human B-lymphoblastoid cell line and macrophage-like cells from neonatal rat brain were also positive for CB1 receptor-like immunoreactivity. Immunocytochemical analysis performed with isolated Fab fragments showed surface staining in NG108-15 cells and brain macrophage like cells which also express MHC class II antigens. The data suggest a plausible role for CB1 receptors in the immune function of brain.

The interaction of hyaluronectin (HN), a hyaluronic acid-binding extracellular matrix (ECM) glycoprotein with two other ECM-associated molecules, laminin and fibronectin, was studied by ligand blot and solid phase ligand binding assays. Ligand blot analysis with biotinlabeled HN revealed a strong binding of HN to immobilized laminin and a weaker binding to fibronectin. Ligand binding studies indicated a concentration dependent, Ca2-independent binding of HN to laminin. Binding of HN to laminin but not to fibronectin was resistant to increased salt concentrations indicating a non-electrostatic, protein-protein interaction of HN with laminin. The functional relevance of HN–laminin interaction was demonstrated by an inhibition of laminin-supported astroglial process formation by HN.

Ewing's sarcoma family of tumors (EFT) contain reciprocal translocations, of which approximately 90% occur between the long arm of chromosomes 11 and 22, t(11;22)(q24;q12), resulting in the formation of chimeric proteins generated by a fusion of the EWS and FLI-1 genes. To determine if EWS-FLI-1 protein is responsible for the Ewing sarcoma phenotype we have used sequence-specific antisense oligodeoxynucleotides (ODN) to block its expression. We have evaluated a series of antisense ODN directed toward the breakpoint region in an effort to prevent translation of the fusion messenger RNA. ODN were first evaluated in a cell-free in vitro translation system. Exogenously added RNase H was found to be required for translation inhibition. ODN that showed complete inhibition of translation were electroporated into TC-32 cells, a EFT cell line. Fusion protein and EWS protein levels were evaluated by Western blot analysis. A 40—60% decrease in the fusion protein was observed in TC-32 cells with antisense ODN directed toward the breakpoint region. Cell viability was reduced with antisense sequences in TC-32 cells but not in a prostate cancer cell line. Since inhibition of t(11;22) gene product is correlated to effects on cell viability, reduction of the fusion protein may thus offer insight into the biology of EFT.

Populations of enriched glial precursor cells and astrocytes isolated from primary cultures of newborn rat brain were used to study the synthesis of sulfated glycoproteins. Both cell types incorporated [3H]glucosamine and [35S]sulfate into carbohydrate side chains of proteoglycans and glycoproteins. The rate of incorporation of [3H]glucosamine into the oligosaccharides and the pattern of distribution of the label into high mannose and complex glycopeptides recovered from the glycoproteins appeared to be similar for the two glial cell types. However, clear differences were noted in the rate of oligosaccharide sulfation activities. Thus the cultures of precursor glia were about four times more active than cultures enriched in astroglia in their ability to incorporate [35S]sulfate into glycoproteins.

We have examined human T-lymphotropic virus type I (HTLV-I) gene expression in the human T-cell line, C8166-45 (C81), as a model to define the gene products expressed from defective proviruses. C81 cells contain one complete and two different deleted proviral genomes. The internal deletions of the latter encompass most of the gag to env region. All three proviruses are transcriptionally active as evidenced by the presence of three unspliced nuclear mRNAs. Unspliced genomic mRNAs and singly spliced env mRNAs were present in the nucleus, but were not detected in the cytoplasm, suggesting a defect in Rex function. Three small cytoplasmic mRNAs were observed and are likely to correspond to the normal 1.8-kb Tax1/Rex1 mRNA, a 1.6-kb mRNA formed by splicing the 5'LTR to the pX region, and a 2.1-kb mRNA of unknown origin. Consistent with the subcellular mRNA distribution pattern, the viral structural proteins encoded by gag and env genes were not detected. The transcriptional transactivator protein, Tax1 (p40), was abundantly expressed in C81 cells; in addition, a 42-kDa Tax1 protein, unique to this cell line, was also detected. Although Tax1 end Rex1 (p27) are translated from overlapping open reading frames in the same mRNA, Rex1 was not detected in C81 cells. The presence of a premature termination codon in the Tax1/Rex1 mRNA encoded by the full-length provirus was inferred from the presence of small Rex1-related polypeptides lacking C-terminal sequences and confirmed by sequence analysis. Furthermore, a p21X protein lacking the N-terminus of Rex1 was expressed at high levels; our data indicate that p21X is translated from the 1.6-kb mRNA which is derived primarily from deleted proviruses. Copyright 1993, 1999 Academic Press

A simple, novel synthesis of (E)-gem-dimetalloalkenes containing boron and silicon based on (Z)-1-bromo-1-alkenyl­boronate esters is developed. 1-Bromo-(Z)-1-alkenylboronate esters readily available from literature procedures smoothly undergo a ­reaction with trimethylsilyllithium (easily generated from hexamethyldisilane with methyllithium in hexamethylphosphoramide) to provide the corresponding ‘ate’ complexes. These ‘ate’ complexes undergo intramolecular nucleophilic substitution reaction to provide the corresponding (E)-1-alkenylboronate esters containing trimethylsilyl moiety in good isolated yields (72-85%). The carbon skeletons present in these intermediates are confirmed by oxidation to the corresponding alkyl trimethylsilyl ketones with sodium ­acetate and hydrogen peroxide.

1-Trimethylgermyl-1-alkynes easily prepared by deprotonation of terminal alkynes with n-butyllithium followed by treatment with trimethylgermanium chloride, readily react with dicyclohexylborane (freshly prepared by reacting cyclohexene with borane-methyl sulfide complex) in tetrahydrofuran. The resulting (Z)-1-trimethylgermyl-1-alkenyl dicyclohexylboranes are readily protonolysed in n-pentane by acetic acid. The monoethanolamine workup, followed by vacuum distillation provides the corresponding (Z)-1-trimethylgermyl-1-alkenes in high stereochemical purities (98%) and in excellent yields (78-92%).

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

For Abstract see ChemInform Abstract in Full Text.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The enzyme nitric oxide synthase 2 (NOS2, or inducible NOS) is expressed by numerous cell types during inflammatory reactions in vivo and in vitro. The NO derived from NOS2 exerts beneficial actions such as the elimination of microorganisms, the reduction of thrombosis, and the increase of blood supply to injured tissues. However, NO released in excess can cause tissue damage, contributing to diseases like septic shock, rheumatoid arthritis, cerebral ischemia, multiple sclerosis, and diabetes. It is well established that induction of the NOS2 protein by inflammatory stimuli is primarily regulated at the level of gene transcription, and that the interaction of the transcription factor NFkB and its inhibitory protein, IkB, play a central role in this process. In addition, recent pharmacological evidence implicates the mitogen activated protein kinase (MAPK) pathways as a second key mediator of the cytokine-induced transcription of NOS2. In turn, the induction of the NOS2 gene can be either inhibited or potentiated, depending on the cell type, by cyclic AMP-dependent pathways, acting via transcription factors like CREB, NFkB and C/EBP. Finally, the heat shock response has recently emerged as a second potent means of suppressing both the cytokine and endotoxin-dependent activation of NOS2 transcription. The mechanism responsible for this suppression appears to be the prevention of NFkB activation. These observations suggest that the transcription of the NOS2 gene is tightly regulated by several cascades of cytoplasmic and nuclear reactions, thus providing a wide array of biological and therapeutical targets to control the production of NO to avoid its detrimental actions.