Rhesus macaques infected with lymphocytic choriomeningitis virus (LCMV) provide a model for human Lassa fever. Disease begins with flu-like symptoms and progresses rapidly with fatal consequences. Previously, we profiled the blood transcriptome of LCMV-infected monkeys  showing distinct pre-viremic and viremic stages that discriminated virulent from benign infections. In the present study, changes in liver gene expression from macaques infected with virulent LCMV-WE were compared to gene expression in uninfected monkeys as well as to monkeys that were infected but not diseased.Based on a functional pathway analysis of differentially expressed genes, virulent LCMV-WE had a broader effect on liver cell function than did infection with non-virulent LCMV-Armstrong. During the first few days after infection, LCMV altered expression of genes associated with energy production, including fatty acid and glucose metabolism. The transcriptome profile resembled that of an organism in starvation: mRNA for acetyl-CoA carboxylase, a key enzyme of fatty acid synthesis was reduced while genes for enzymes in gluconeogenesis were up-regulated. Expression was also altered for genes associated with complement and coagulation cascades, and with signaling pathways involving STAT1 and TGF-β.Most of the 4500 differentially expressed transcripts represented a general response to both virulent and mild infections. However, approximately 250 of these transcripts had significantly different expression in virulent infections as compared to mild infections, with approximately 30 of these being differentially regulated during the pre-viremic stage of infection. The genes that are expressed early and differently in mild and virulent disease are potential biomarkers for prognosis and triage of acute viral disease. Arenaviruses are rodent-borne viruses that can be transmitted to primates, occasionally causing lethal hemorrhagic fever. Arenaviruses causing Lassa fever and South American hemorrhagic fevers have been classified as Category A bio-threats in the United States because of their virulence. Human beings infected with a hemorrhagic fever virus initially exhibit flu-like symptoms, and disease progresses so rapidly that diagnosis and appropriate treatments are often too late. Laboratory studies using the arenavirus lymphocytic choriomeningitis virus (strain LCMV-WE) showed that rhesus macaques develop an acute viral disease similar to Lassa fever in human beings -8. LCMV-Our previous publications on the pathology of LCMV-WE infection described up-regulation of liver gene expression related to organ development, regeneration and inflammatory responses ,3,5. Blocaveats in mind, we examine the most prominent transcriptome changes in relation to published information about primate liver infections. Down-regulated genes involved in fatty acid synthesis and up-regulated genes involved in gluconeogenesis presented a profile that has been associated with starvation and also typifies LCMV infection of macaques. Although most of the gene expression changes controlling intermediary metabolism could be categorized as general homeostatic responses to infection, some gene-expression changes, such as in transcripts related to amino-acid catabolism and protein phosphatase, were strongly associated with an early virulent profile and were more likely to contribute to fatal disease. Approximately 30 genes were identified as potential signature biomarkers for the onset of virulent LCMV-related liver disease. We discuss those gene expression changes that are similar to other viral diseases of liver and those changes that seem unique to an acute arenavirus infection.Despite the complex clinical presentation of viral hemorrhagic fever, we chose to focus on liver gene expression because that organ had the highest virus titers. Liver tissue contains several cell types, and approximately 25% of the changes in transcriptome do not result in proteomic changes ; so withTwenty healthy adult rhesus macaques, five to nine years of age, were used for a terminal study . Liver t3 plaque forming units (pfu) virus. LCMV-WE alone, at 103 pfu i.v. is uniformly lethal. Four animals infected with both LCMV-ARM and LCMV-WE (103 pfu each), did not develop symptoms and were classified as "infected-but-not-diseased". Infection in macaques was monitored by plaque assay of infectious particles in plasma, by infectious center assay of PBMC and liver tissues, and by RT-PCR to detect viral RNA in tissues . Differentially-regulated genes were selected using a 2-fold cut-off and a false discovery rate (FDR) of < 0.05.A total of 20 samples were used to generate the microarray data E) Table . ClusterE) Table . Mean n-n-fold of at least 2 and FDR P < 0.05) between infected samples (n = 9) and uninfected control samples (n = 3) or between infected samples (n = 9) and samples from animals that were infected but not diseased (n = 6). Six separate pair-wise comparisons were also performed to compare the three groups of infected samples: pre-viremic (n = 4), viremic (n = 5) and post-viremic (n = 2) to the two groups; controls (n = 3) or non-diseased samples (n = 6). These six pairwise comparisons resulted in identification of 4,482 differentially-regulated genes (n-fold of at least 2 and FDR P < 0.05) amongst any one pairwise comparison. KEGG software was used to identify key functions and metabolic pathways differentially-regulated between uninfected and LCMV-infected liver samples. The raw gene expression data and the related experimental information from this study can be found at , platform number GSE12254.Pairwise comparisons were performed to identify 3,125 differentially-regulated genes .Genes for microarray analysis were validated by quantitative real-time PCR as we described  to deterLiver RNA from LCMV-infected macaques was used for transcriptome profiling with the goal of identifying gene expression that differentiated infected from uninfected liver and that differentiated virulently-infected (diseased) from mildly-infected (non-diseased) liver. As described previously , we charThe liver is highly vascularized and sensitive to changes in blood composition. Since liver tissue contains a mixture of cells including hematopoietic cells found in the circulation, we examined the overlap of gene expression in liver and in blood for the virulently-infected monkeys Figure , 5, 6, 7p < 0.05) up-regulated genes and 123 down-regulated genes (the remainder were up-regulated at some time points and down-regulated at others). Differential gene expression is presented in two ways: 1) LCMV-WE-infected (diseased) liver compared to uninfected liver, and 2) LCMV-WE-infected (diseased) liver compared to LCMV-infected non-diseased liver. Differentially-expressed genes were defined as those that had at least a 2-fold alteration in LCMV-infected livers compared to the uninfected controls.Additional File 53 are listed in Figure Genes with the highest differential expression between LCMV-infected and uninfected livers , were up-regulated during LCMV infection. Nevertheless, the liver-specific transporter gene APOAV was down-regulated  in both pre-viremic and viremic stages of infection Table  suggestiMobilization of fat (lipolysis) is favored under conditions of increased energy need, such as exercise, fasting, hypothermia , or cachFree fatty acids (FFA) stimulate key gluconeogenic enzymes ,29. For The aldehyde dehydrogenases clear the blood of toxic aldehydes by generating acetate, which can be converted to acetyl CoA. Several aldehyde dehydrogenases are up-regulated during infection, especially ALDH6A1, a liver mitochondrial enzyme that catalyzes the breakdown of malonate to propionyl – and acetyl-CoA,  occurs during high rates of fatty acid oxidation through generation of large amounts of acetyl-CoA in liver . Ketone The expression of several lipase genes is strongly up-regulated during LCMV-WE infection, suggesting a relationship between up-regulation of beta-oxidation enzymes and lipolysis in the infected livers Table . In geneThe complement and coagulation cascades interact and modulate each other; both are affected during LCMV infection. Although we do not present coagulation data here, platelet defects and a mild thrombocytopenia were documented during severe Lassa fever ,12, and Genes that discriminate between virulent and non-virulent infection, especially if they do so during the pre-viremic stage, have the greatest potential as prognostic biomarkers for severe arenaviral disease. Virulent and non-virulent liver transcriptomes were compared by pooling results from nine animals (LCMV-WE-infected) to compare with six samples from animals that were LCMV-infected but not diseased . In our study, the transcriptome of non-diseased animals closely resembled the transcriptome of animals infected with non-virulent LCMV-ARM , serine dehydratase (SDS), and glutamic-oxaloacetic transaminase (GOT1), were not altered in their expression when comparing infected versus uninfected samples, yet there was a significant down-regulation of these genes when comparing LCMV-WE-infected to LCMV-non-diseased samples Figure . This me, serine PPP1R3C is the most dramatically up-regulated transcript in virulently-infected livers with respect to mildly-infected liver samples (156-fold in the pre-viremic stage), and this up-regulation has been validated by quantitative PCR Table . PPP1R3CAdditional notable differences between virulent and mild infections are large increases in expression of genes for activin receptor (ACVR1) and inhibin , along with a decrease in expression of follistatin (FST); all of which belong to the transforming growth factor beta (TGF-β) gene family. Expression of these TGF-β family genes was validated by real-time quantitative PCR Table . Though The LCMV-infected monkey model for Lassa fever was investigated with the goal of finding transcriptome changes that could eventually be used as prognostic biomarkers to discriminate virulent and mild infections. Transcriptome analysis of rhesus macaque liver showed that both mild and virulent LCMV infections had tremendous effects on glucose, amino acid and fatty acid metabolism, and on the complement and coagulation cascades. As with our previous transcriptome analysis using blood , promineA transcriptome study using the HuH-7 liver cell line  showed tOther liver transcriptome studies have identified major molecular events associated with viral infection, with higher levels of interferon-responsive genes being a common theme. We observed significant up-regulation of ISG15, IFIT1, and STAT1 in LCMV-WE-infected (diseased) monkey liver with respect to non-diseased liver, and similar increased expression was seen in human liver infected with hepatitis C virus . As noteTwo crucial liver functions are glycogen breakdown and gluconeogenesis from non-sugars like fatty acids, pyruvate, lactate, and amino acids, with both functions beginning a few hours after the onset of fasting. Elevated rates of fat breakdown (lipolysis) increase the release of free fatty acids that simultaneously stimulate gluconeogenesis and inhibit glycogenolysis ,50. The Other viral infections are also inter-twined with glucose, amino acid and fatty-acid metabolism. The transcription of hepatitis B virus, a DNA virus, requires a key regulatory enzyme of gluconeogenesis, peroxisome proliferator-activated receptor γ C1 (PPARGC1. This enThe acute LCMV disease has characteristics of starvation that resemble both cachexia (TNF-α or IL-6-mediated wasting) and anorexia (appetite loss). High levels of IL-6 were detected in LCMV-WE-infected liver , whereasIt is difficult to relate changes in intermediary metabolism of LCMV-infected liver to a lethal disease that resembles Lassa fever. Although the liver transcriptome may resemble that of a starving organism, and such a profile is supported by the dehydration and appetite-loss observed in diseased monkeys, it is known that primates can survive weeks of starvation ; so it i6 pfu virus, whereas the monkeys were given 103 pfu. Also, the disease in mice is generally considered immunopathological, i.e. alleviated by immunosuppression, unlike the disease in guinea pigs and primates [Some of the differences between virulently – and mildly-infected liver could be ascribed to the over-expression of interferon-response genes  in blood, but not in liver.Click here for fileLists genes from the Venn diagram of Figure 1in the form of 500 probe sets. "Pre" means they were expressed in the pre-viremic stage (day 1–3) in both blood and liver.Click here for fileLists genes from the Venn diagram of Figure 1in the form of 2033 probe sets. "Pre" means they were expressed in the pre-viremic stage (day 1–3) in liver, but not in blood.Click here for fileLists genes from the Venn diagram of Figure 1 in the form of 3136 probe sets. "Vir" means they were expressed in the viremic stage (day 4–7) in blood, but not in liver.Click here for fileLists genes from the Venn diagram of Figure 1in the form of 963 probe sets. "Vir" means they were expressed in the viremic stage (day 4–7) in blood and liver.Click here for fileLists genes from the Venn diagram of Figure 1in the form of 1722 probe sets. "Vir" means they were expressed in the viremic stage (day 4–7) in liver, but not in blood.Click here for fileList of differentially-expressed genes in LCMV-infected liver. This list of human probe sets identified in hybridization of rhesus macaque liver cRNA includes those transcripts differentially expressed with respect to uninfected (uninf) control samples (n = 4484), or those transcripts differentially expressed with respect to samples from monkeys that were LCMV-infected but not diseased (nodis). These were identified as differentially expressed using LIMMA analysis. WE1 refers to 4 liver samples taken day 1 to day 3 after infection (pre-viremic stage). WE2 refers to 5 liver samples taken day 4 to day 7 after infection (viremic stage), and WE3 refers to 2 liver samples taken day 11 and day 12 after infection . WEx vs uninf refers to virulent samples being compared to data from 3 uninfected samples. WEx vs nodis refers to virulent samples being compared to data from 6 infected but not diseases samples. Samples are more fully described in Table p-values from LIMMA. Significantly regulated genes were selected using a 2-fold cut-off and a false discovery rate of < 0.05.Click here for filePathway gene expression in LCMV-WE-infected macaque liver tissues. Significantly affected pathway gene expression in LCMV-WE-infected macaque liver tissues. KEGG software was used to identify groupings of genes with pathways based on published data. False discovery rate of the pair-wise comparisons were calculated using p-values from LIMMA. Significantly regulated genes were selected using a 2-fold cut-off and a false discovery rate of < 0.05.Click here for file


Three populations active in human spontaneous cytotoxicity have been identified. Two of these are E-rosette positive, and differ in their adherence to nylon wool. The third is E-rosette negative. The E-rosette positive fraction which does not adhere to nylon consistently does not lyse a breast-cancer-derived target, MDA-157. When tested simultaneously on 4 other tumour target cells lines--Raji, Chang, K562 and Molt 4--however, all three populations are cytolytic. The MDA-157 target is consistently lysed by a nylon-adherent T-cell fraction, irrespective of whether the E rosettes are formed under optimal or the limiting conditions giving only "high-affinity" T cells. The observation that a given effector fraction can lyse one target but not another, whereas other fractions are cytolytic on both, implies that different targets may differentiate effector populations differing in their lytic mechanism.


The human multidrug resistance protein (MRP1) confers resistance of cells to a number of different cytostatic drugs and functions as an export pump for glutathione S-conjugates, glucuronides and other amphiphilic anions. The present study details for the first time MRP1-mediated ATP-dependent transport of various glutathione S-conjugates of the bifunctional alkylating agents chlorambucil and melphalan. In membrane vesicles prepared from cells expressing recombinant MRP1, the conjugates were transported at rates in the following order: monoglutathionyl chlorambucil > bisglutathionyl chlorambucil > monohydroxy monoglutathionyl chlorambucil and monoglutathionyl melphalan > monohydroxy monoglutathionyl melphalan. In addition, we show that membranes from chlorambucil-resistant GST-alpha-overexpressing CHO cells as well as from their parental cells express the hamster homologue of MRP1. With both CHO cell membrane preparations, we observed ATP-dependent transport of monoglutathionyl chlorambucil and of leukotriene C4, a glutathione S-conjugate and high-affinity substrate of MRP1. The transport rates measured in the resistant cells were only two- to three-fold higher than those measured in the control cells. These results together with cytotoxicity assays comparing MRP1-overexpressing cell pairs with the CHO cell pair indicate that, although MRP1-mediated transport is active, it may not be the rate-limiting step in chlorambucil resistance in these cell lines.


Deregulated lipogenesis plays an important role in tumour cell survival. These observations suggest that enzymes in the lipid synthesis pathway would be rational therapeutic targets for cancer. To this end, we review the enzymes in  ACC carboxylates acetyl-CoA to form malonyl-CoA. The malonyl-CoA product is further converted by FASN to long-chain fatty acids. Most normal human tissues preferentially use dietary (exogenous) lipid for synthesis of new structural lipids, whereas de novo (endogenous) fatty-acid synthesis is usually suppressed, and FASN expression is maintained at low levels. By contrast, in cancer cells, de novo fatty-acid synthesis is commonly elevated and the supply of cellular fatty acid is highly dependent on the de novo synthesis. Therefore, deregulated de novo fatty-acid synthesis directly leads to cellular fatty-acid accumulation and affects fundamental cellular processes, including signal transduction and gene expression. Numerous studies have shown overexpression of FASN in various human epithelial cancers, including prostate, ovary, colon, lung, endometrium and stomach cancers  and HER2, and steroid hormone – steroid hormone receptors, such as oestrogen receptor (ER), androgen receptor (AR) and progesterone receptor (PR). Downstream of the receptors, the phosphatidylinositol-3-kinase (PI3K)-Akt and mitogen-activated protein kinase (MAPK) are candidate signalling pathways that mediate FASN expression through the sterol regulatory element-binding protein 1c (SREBP-1c). However, the regulations of FASN expression by the growth factor receptors are not simple. For instance, mutual regulation between FASN and the growth factor-dependent signalling is suggested. HER2 induces FASN expression through the downstream PI3K signalling  interacts with and stabilises FASN protein in prostate cancer -2-furoic acid does not  catalyses the conversion of citrate to cytosolic acetyl-CoA, thus linking the tumour-associated increase in glycolysis to enhanced lipogenesis . As withβ-oxidation, and lipid modification of cellular proteins  is an enzyme that acts downstream of FASN and converts long-chain fatty acids to acyl-CoA . This rede novo fatty-acid synthesis has been recognised as a hallmark of cancer, whereas its significance in cancer pathogenesis has long been underestimated. However, the recent advances in this field have shown the importance of the lipogenic enzymes in tumour cell survival. From the viewpoint of cancer prevention, it would also be important to take into account the effects of dietary factors, such as conjugated linoleic acid, on lipid metabolism in carcinogenesis (Increased


We previously demonstrated that overexpression of Golgi-localized, γ-ear containing, Arf-binding (GGA) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs). GGA overexpression led to the formation of large, swollen vacuolar compartments, which in the case of GGA1 sequestered HIV-1 Gag.In the current study, we extend our previous findings to characterize in depth the GGA-induced compartments and the determinants for retroviral Gag sequestration in these structures. We find that GGA-induced structures are derived from the Golgi and contain aggresome markers. GGA overexpression leads to defects in trafficking of transferrin receptor and recycling of cation-dependent mannose 6-phosphate receptor. Additionally, we find that compartments induced by GGA overexpression sequester Tsg101, poly-ubiquitin, and, in the case of GGA3, Hrs. Interestingly, brefeldin A treatment, which leads to the dissociation of endogenous GGAs from membranes, does not dissociate the GGA-induced compartments. GGA mutants that are defective in Arf binding and hence association with membranes also induce the formation of GGA-induced structures. Overexpression of ubiquitin reverses the formation of GGA-induced structures and partially rescues HIV-1 particle production. We found that in addition to HIV-1 Gag, equine infectious anemia virus Gag is also sequestered in GGA1-induced structures. The determinants in Gag responsible for sequestration map to the matrix domain, and recruitment to these structures is dependent on Gag membrane binding.These data provide insights into the composition of structures induced by GGA overexpression and their ability to disrupt endosomal sorting and retroviral particle production. The Gag polyprotein precursors are the key structural elements driving retroviral particle production. The N-terminal matrix (MA) domain of the Gag precursor is important for plasma membrane (PM) targeting and membrane binding. Following Gag targeting, the process of assembly proceeds via Gag multimerization mediated primarily by sequences within the capsid (CA) and nucleocapsid (NC) domains. Finally, the "late" domains in Gag mediate the terminal step in particle production - the pinching off of the virion from the infected cell membrane -5. ConcoGag, contains in its p6 domain a Pro-Thr/Ser-Ala-Pro [P(T/S)AP] motif that binds Tsg101, a component of the endosomal sorting complex required for transport-I (ESCRT-I)  is a key cellular cofactor for HIV-1 Gag targeting to the PM . Th. Thcis cTreatment of cells with the fungal toxin brefeldin A (BFA) leads to the dissociation of Arf-dependent coats, including the GGA proteins, from membranes ,52,53. IWe next characterized the GGA-induced compartments by determining whether they are aggresome-like structures. Aggresomes are formed in response to protein misfolding or high protein concentrations in the cytoplasm . Other fAs shown above, GGA overexpression leads to the accumulation of large swollen compartments. We hence investigated the physiological consequences of GGA overexpression by monitoring epidermal growth factor (EGF) receptor internalization, TfR endocytosis, and CD-MPR recycling. GGA-overexpressing cells were treated with Texas Red-tagged EGF or fluorescently tagged transferrin (Tf-594) conjugates in serum free medium, followed by fluorescence microscopy. GGA overexpression did not significantly alter EGF receptor internalization (data not shown). However, GGA overexpression led to abnormal uptake of transferrin conjugates from the PM. As shown in Figure We next studied the effect of GGA overexpression on the localization of host factors described to be important for retrovirus release. To this end, cells were transfected with vectors expressing HA-tagged Tsg101 and Myc-tagged GGA1, 2, or 3 and analyzed by fluorescence microscopy. Exogenously expressed, full-length Tsg101 (TSG-F) localized to enlarged endosomal structures throughout the cell Figure .To further define the composition of the GGA-induced structures, we next asked whether they sequester ubiquitin or ubiquitinated cargo. HeLa cells transfected with GGA expression vectors were stained with an antibody that recognizes polyubiquitinated proteins but not free ubiquitin . As showWe demonstrated previously that GGA overexpression inhibits retrovirus particle production, which in the case of GGA1 is in part due to sequestration of HIV-1 Gag in GGA1-induced structures . We alsoPreviously we showed that GGA overexpression modulates retrovirus assembly by inhibiting Gag targeting to the PM. GGA overexpression also induced the accumulation of large vacuolar structures, which in the case of GGA1 sequestered HIV-1 Gag . In the GGA proteins when expressed exogenously in cells whether transiently or stably have been reported to show a localization pattern similar to that of their endogenous counterparts ,49. IndeGGA overexpression also led to alterations in the subcellular localization of several host proteins implicated in retrovirus assembly and release. We previously observed that GGA overexpression led to sequestration of Arfs . MoreoveAlthough the three mammalian GGA proteins are closely related, in our studies they have shown some notable differences: 1) depletion of GGA3, and to a lesser extent GGA2, stimulated HIV-1 release whereas GGA1 depletion did not . 2) WherIn contrast to the Gag sequestration data, in virus release assays both Fyn10FullMA  and Fyn10deltaMA  were resIn the current study, we extend our previous findings and characterize in detail the structures induced by GGA overexpression with respect to their impact on cellular sorting pathways and their ability to inhibit retroviral particle production. We observe that the compartments induced by GGA overexpression are distinct from those formed by dominant-negative components of the ESCRT pathway, both in terms of their effect on cellular functions and with respect to the step in the retrovirus assembly and release pathway that they disrupt. A better understanding of these host components will not only further our knowledge of cellular sorting processes but also help define the requirements for host factors in retrovirus assembly and release.HeLa cells were cultured in DMEM supplemented with 5% fetal bovine serum (FBS) and 2 mM glutamine. All transfections were performed using Lipofactamine2000™ reagent (Invitrogen) as per the manufacturer's instructions.Plasmids expressing full-length Myc-tagged GGA proteins were kindly provided by J. Bonifacino  ,48,49. TImmunostaining of cells was performed as described , with mi24 h posttransfection, cells were washed three times with serum free medium (SFM) containing 20 mM HEPES and 1% BSA and incubated for 1 h at 37°C in the same medium. Cells were then placed on ice for 5 min followed by incubation for 30 min at 4°C in SFM containing 5 μg/ml EGF-Texas Red or 10 μg/ml Tf-594 (Molecular Probes). Cells were washed three times with PBS and incubated in cell culture medium at 37°C for 15 min for TfR and 30 min for EGF receptor. Finally, cells were washed with PBS, fixed and mounted using DAPI-containing medium for EGF receptor assays.35S]Met/Cys. Culture supernatants were ultracentrifuged at 100,000 × g for 45 min, cell and virus lysates were immunoprecipated with HIV-Ig, resolved by SDS-PAGE followed by PhosphorImager analysis. Virus release was calculated as the percentage of virion-associated p24 (CA) relative to total (virion + cell-associated) Gag. Virus release efficiency = virion p24/(cell associated Pr55Gag + cell-associated p24+virion-associated p24) × 100.The protocol for radiolabeling and immunoprecipitation of cell and virus lysates has been described in detail previously . Briefly2: phosphatidylinositol--bisphosphate; PM: plasma membrane; POSH: plenty of SH-3s; PR: protease; P(T/S)AP: Pro-Thr/Ser-Ala-Pro; RSV: Rous sarcoma virus; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; SFM: serum-free medium; STAM: signal transducing adaptor molecule; TfR: transferrin receptor; TGN: trans-Golgi network; Tsg101: tumor susceptibility gene 101; SOCS1: suppressor of cytokine signaling 1; VHS: Vps27, Hrs and STAM homology; VLP: virus-like particle.Alix: ALG-2 interacting protein X; AP: adaptor proteins; Arfs: ADP ribosylation factors; BFA: brefeldinA; BSA: bovine serum albumin; CA: capsid; CD-MPR; cation-dependent mannose 6-phosphate receptor; DMEM: Dulbecco-modified Eagle's medium; EEA: early endosomal antigen; EGF: epidermal growth factor; EIAV: equine infectious anemia virus; EM: electron microscopy; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FBS: fetal bovine serum; FIV: feline immunodeficiency virus; GAE: γ-adaptin ear homology domain; GAT: GGA and Tom domain; GGA: Golgi-localized, γ-ear containing, Arf-binding; GM130: Golgi matrix marker 130; Hrs: hepatocyte growth factor-regulated tyrosine kinase; MA: matrix; MLV: murine leukemia virus; MVB: multivesicular body; NC: nucleocapsid; PBS: phosphate-buffered saline; PIPAJ performed the experiments and participated in the design of experiments, the interpretation of the data, and the writing of the manuscript. KN performed EM analysis. EOF participated in the design of experiments, the interpretation of the data, and the writing of the manuscript. All authors read and approved the final version of the manuscript.


Therapy with human lymphoblastoid interferon HuIFN-alpha(N1), or recombinant human interferon gamma, rHuIFN-gamma, inhibited experimental pulmonary metastases of the human melanoma cell line, DX3-azac, in BALB/c nude mice and significantly prolonged survival. The human IFNs had no effect on nude mouse lung and spleen NK cell activity, lung macrophage activity, haemoglobin or white cell counts. HuIFN-alpha(N1) had no effect on the levels of the IFN induced enzyme 2-5A synthetase in nude mouse lungs although the rHuIFN-gamma caused some elevation. In addition, clearance of radiolabelled DX3-azac cells was identical in control or human IFN treated mice, and there was no histological evidence of an increase in immune effector cells associated with the metastatic lesions in treated mice. Human IFN therapy did not affect the state of differentiation of the melanoma cells in vivo as measured by melanin content, but both IFNs inhibited the development of colonies of DX3-azac cells in vitro. We conclude that in this model system IFNs have direct anti-proliferative effects on metastatic cells.


P. falciparum (Pf) accumulates hemozoin (HZ), the crystal of dimerized undigested heme. HZ wrapped up in the food vacuole membrane binds saturated and polyunsaturated fatty acids (PUFA) and proteins, generates bioactive ligands and is expelled in the blood during schizogony as residual body. HZ contains redox active iron that peroxidizes PUFA to produce bioactive hydroxy-PUFA  and terminal aldehydes . HETEs is responsible for some of the HZ effects: (1) down-modulation of oxidative burst; (2) inhibition of dendritic cells (DC) differentiation-maturation; and (3) enhancement of expression and enzyme activity of metalloproteinase MMP-9. Low-micromolar HETE recapitulated effects (2) and (3). Recapitulation was abrogated by PPAR-gamma-receptor inhibitors.Blood stage Other effects, such as inhibition of erythropoiesis, were due to the transfer of HNE to differentiating human erythroid precursors. Erythropoiesis was inhibited by transferred HNE via blockage of cell cycle and the down-regulation of protein expression of crucial receptors . Malarial dyserythropoiesis, which plays an important role in malaria anaemia (MA) is characterized by (1) decreased growth and differentiation of erythroid precursors; (2) low reticulocyte response; and (3) normal production of erythropoietin (EPO). In human bone marrow (BM) the erythroblastic island (EI) is the elementary unit of erythropoiesis in which a central macrophage (MAC) is surrounded by differentiating erythroid cells. In malaria anaemia, HZ-laden central MACs are closely adjacent to developing erythroid cells. HZ and HZ-laden human monocytes inhibited growth of co-cultivated human erythroid cells and produced HNE that diffused to adjacent cells generating HNE-protein adducts. Co-cultivation with HZ or treatment with low-micromolar HNE inhibited growth of erythroid cells interfering with cell cycle without apoptosis. Following HZ/HNE treatment, two critical proteins in cell cycle regulation, p53 and p21, were increased and the retinoblastoma protein, central regulator of G1-to-S-phase transition, was consequently hypophosphorylated, while GATA-1, master transcription factor in erythropoiesis was reduced. The resul was decreased expression of cyclin A and D2 retarded cell cycle progression in erythroid cells and the K562 cell line. As a second major effect, HZ and HNE inhibited protein expression of crucial receptors (R): transferrin R1, stem cell factorR, interleukin-3R and erythropoietinR. The reduced receptor expression and the impaired cell cycle activity decreased the production of cells expressing glycophorin-A and hemoglobin.In conclusion, present data confirm the inhibitory role of HZ, identify HNE as one HZ-generated inhibitory molecule and describe molecular targets of HNE in erythroid progenitors possibly involved in erythropoiesis inhibition in malaria anemia.Supported by Regione Piemonte, IARC and Compagnia di San Paolo grants to ES, WP and PA.


Multiple myeloma is often associated with humoral immunodepression in both man and mouse. When mice bearing the humorally immunodepressive plasmacytomas TEPC-183 and SPQC-11 were injected with SRBC, the rise of serum haemolysins was significantly less than that of non-tumour-bearing mice. Mice with the plasmacytomas MPC-11 and MOPC-315 have an antibody response similar to normal mice when injected with SRBC. Following immunization, normal mice and those bearing MPC-11 showed a 2- to 3-fold increase in total spleen lymphocytes. Mice bearing TEPC-183 or SPQC-11, the plasmacytomas causing an impaired antibody response, has significant increase in spleen lymphocytes under the same conditions. Mice bearing MOPC-315 had a very high initial count of spleen lymphocytes, which did not further increase upon immune stimulation.Incubation of lymphocytes from plasmacytoma-bearing mice with PHA did not produce an increase in TdR incorporation and in some cases even caused a decrease in TdR incorporation.in vitro correlated with the lack of an increase of spleen lymphocytes in mice bearing these tumours following antigenic stimulation in vivo.Lymphocytes from mice bearing TEPC-183, SPQC-11, and MOPC-315 incorporated less TdR in response to LPS than did normal mice. On the other hand, mice bearing MPC-11 incorporated about as much TdR as did normal mice following LPS stimulation. Thus, the defect in the ability to respond to LPS No immunodepressive properties of serum from mice with plasmacytoma could be detected.


Caveolin-1                        (-/-) null stromal cells are a novel genetic model for cancer-associated                        fibroblasts and myofibroblasts. Here, we used an unbiased informatics                        analysis of transcriptional gene profiling to show that Cav-1 (-/-)                        bone-marrow derived stromal cells bear a striking resemblance to the                        activated tumor stroma of human breast cancers. More specifically, the                        transcriptional profiles of Cav-1 (-/-) stromal cells were most closely                        related to the primary tumor stroma of breast cancer patients that had                        undergone lymph-node (LN) metastasis. This is consistent with previous                        morphological data demonstrating that a loss of stromal Cav-1 protein  is                        significantly associated with increased LN-metastasis. We also provide                        evidence that the tumor stroma of human breast cancers shows a                        transcriptional shift towards oxidative stress, DNA damage/repair,                        inflammation, hypoxia, and aerobic glycolysis, consistent with the "Reverse                        Warburg Effect".  Finally, the tumor stroma of "metastasis-prone" breast                        cancer patients was most closely related to the transcriptional profiles                        derived from the brains of patients with Alzheimer's disease. This suggests                        that certain fundamental biological processes are common to both an activated                        tumor stroma and neuro-degenerative stress.  These processes may include oxidative                        stress, NO over-production (peroxynitrite formation), inflammation, hypoxia, and                        mitochondrial dysfunction, which are thought to occur in Alzheimer's disease                        pathology.  Thus, a loss of Cav-1 expression in cancer-associated myofibroblasts                        may be a protein biomarker for oxidative stress, aerobic glycolysis, and                        inflammation, driving the "Reverse                        Warburg Effect" in the tumor micro-environment                        and cancer cell metastasis.                  Recently, we identified a loss of stromal caveolin-1                        (Cav-1) as a novel biomarker for the cancer-associated fibroblast phenotype in                        human breast cancers .       To determine if loss of stromal Cav-1 has prognostic                        value, we performed a series of independent biomarker studies ,4.  UsinA loss of stromal Cav-1 also appears to                        play a role in tumor initiation and progression . Using aTo begin to understand the mechanism(s) underlying the                        lethality of a loss of Cav-1 in cancer-associated fibroblasts, we turned to                        Cav-1 (-/-) deficient mice as a model system.                                For this purpose, we isolated bone marrow derived                        stromal cells from WT and Cav-1 (-/-) deficient mice, as cancer-associated                        fibroblasts are thought to evolve from mesenchymal stem cells . These cIn order to determine which transcriptional programs                        are activated in Cav-1 (-/-) stromal cells, we performed an extensive                        bioinformatics analysis of our genome-wide profiling data . This inHere, we have used a bioinformatics approach to                        determine whether similar "Warburg-like" transcrip-tional profiles exist in the                        tumor stroma isolated from human breast cancers.  For this purpose, we analyzed                        an existing data set in which the tumor stroma was isolated away from adjacent                        breast cancer cells using laser-capture micro-dissection .  We nowFinally, the "Reverse Warburg Effect" is strikingly                        similar to the theory of "Neuon-Glia Metabolic Coupling" -18, whicAs such, tumors may be initiating a                        survival mechanism that is normally used by the brain during stress.                        Interestingly, myofibroblasts and mesenchymal stem cells are known to often                        express GFAP  -21, an iPreviously, we subjected                            Cav-1 (-/-) bone marrow derived stromal cells, and their wild-type                            counter-parts to genome-wide transcriptional profiling .  BecausTo test this hypothesis                            directly, we obtained the transcriptional profiles of a large data set of human                            breast cancer patients  whose tuTumor Stroma vs.                                    Normal Stroma List                    - Compares the transcriptional profiles of tumor stroma                            obtained 53 patients to normal stroma obtained from 38 patients. Genes                            transcripts that were consistently upregulated in tumor stroma were selected                            and assigned a p-value, with a cut-off of p <0.05  (Supplementary                            Table 1) Recurrence Stroma List                    -                            Compares the transcript-tional profiles of tumor stroma obtained from 11 patients                            with tumor recurrence to the tumor stroma of 42 patients without tumor                            recurrence. Genes transcripts that were consistently upregulated in the tumor                            stroma of patients with recurrence were selected and assigned a p-value, with a                            cut-off of p <0.05  (Supplementary Table 2) Lymph-node (LN) Metastasis Stroma List                    - Compares the transcriptional                            profiles of tumor stroma obtained from 25 patients with LN metastasis to the                            tumor stroma of 25 patients without LN metastasis. Genes transcripts that were                            consistently upregulated in the tumor stroma of patients with LN metastasis                            were selected and assigned a p-value, with a cut-off of p <0.05  (                    Supplementary Table 3)-3).  Also, more than 1,000 gene transcripts                            were common between the Cav-1 (-/-) stromal gene list and the gene transcripts                            upregulated in the breast cancer tumor stroma of patients with tumor recurrence                            (p = 1 x 10-3). Finally, nearly 500 genes were commonly upregulated                            between Cav-1 (-/-) stromal cells and the breast cancer tumor stroma of                            patients with LN metastasis (p = 4.6 x 10-6).  Thus, the                            transcriptional profiles of Cav-1 (-/-) stromal cells are most  ignificantly                            related to the tumor stroma of patients with LN-metastasis. Independently, our                            previous data demonstrated that a loss of stromal Cav-1 protein expression (by                            immuno-histochemistry) in human breast cancers is specifically associated with                            a 2.6-fold increase in the number of tumor cell positive lymph nodes                            (LN-metastasis) [These                            three gene lists were then individually intersected with the transcriptional                            profile of Cav-1 (-/-) null stromal cells . The resasis p = .6 x 10-6The top 100 most significant gene transcripts for all                            three human breast cancer stromal gene lists, including their transcriptional                            intersection with Cav-1 (-/-) stromal cells, is included in Supplementary                            Tables 3, 4, and 5.                                        As Cav-1 (-/-) stromal cells are a genetic model of                            activated myofibroblasts  which biCav-1                            (-/-) stromal cells have also been previously subjected to extensive analysis                            via an unbiased proteomics approach ,24.   WeRecently,                            based on the unbiased proteomic and transcriptional analysis of Cav-1 (-/-)                            stromal cells, we have proposed that tumor stromal fibroblasts may undergo                            aerobic glycolysis . We haveTranscriptional                            analysis of Cav-1 (-/-) stromal cells  indicateTable It is important to note that these breast cancer stromal gene lists also include                            Cxcl12, a known HIF-target gene , that isWe have previously shown that the transcriptional profiles of Cav-1 (-/-) stromal                            cells significantly ovelap with the transcriptional profiles obtained from the                            analysis of Alzheimers disease brain . We beliThus, here, we independently evaluated the association                            between Alzheimer's disease and human breast cancer tumor stroma. These                            transcriptional overlaps are enumerated in Table Interestingly, as predicted, the genes that are                            transcriptionally upregulated in Alzheimer's disease significantly overlap with                            tumor stroma, "recurrence-prone" stroma, and "metastasis-prone" stroma. This                            clearly functionally links Alzheimer's disease with the human breast cancer                            tumor stroma.                                        -5).                                        As with the gene profiles of Cav-1 (-/-) stromal                            cells, the Alzheimer's disease profiles were most significantly associated with the "metastasis-prone" stromal gene                            set  stromal cells , consist-10; BRCA_ER_NEG [>                    2.0                             (KO/WT) and a P value of <                    0.1 were utilized for comparison with                            existing gene sets in the data base.                                        A comparison of the Cav-1 (-/-) stromal cell gene set with other existing                            transcriptional profiles also shows significant overlap with ER-negative human                            breast cancer (p = 8.96 x 10A_ER_NEG ). For th>                    1.5;                            p <                    0.05). See also Supplementary Tables.                                        Interestingly, these tumors                            were not laser-capture micro-dissected, so this provides an indication that the                            Cav-1 (-/-) stromal gene set may also be well represented in the                            transcriptional profiles obtained from whole tumors. A HeatMap containing these                            intersecting genes is shown in Figure metabolism and glycolysis                            , hypoxia (Hyou1), the inflammatory response                            , myofibroblast differentiation and                                            the extracellular matrix , as well as DNA-damage and repair                            .  These results are consistent with the existence of the                            "Reverse Warburg Effect" in ER-negative breast cancers.                                        These include key                            overlapping genes associated with Interestingly, it has been previously demonstrated that key secreted inflammatory factors, such as Aif1                             (upregulated nearly 3-fold in Cav-1 (-/-)                            stromal cells; Supplementary Tables) promote NFkB-activation, the paracrine                            growth of ER-negative breast cancer cells , and are-24).                                        Similarly, Aif1 expression                            is highly-upregulated in the tumor stroma of human breast cancers  stromal cells bear a striking resemblance                        to the activated tumor stroma of human breast cancers. More specifically, the                        transcriptional profiles of Cav-1 (-/-) stromal cells were most closely related                        to the stroma of breast cancer patients that had undergone LN-metastasis. This                        is consistent with our previous data showing that a loss of stromal Cav-1                        protein expression (by immuno-histochemistry) in human breast cancer tumor                        micro-arrays is specifically associated with increased LN-metastasis ,4.     Moreover, we provide                        evidence that the tumor stroma of human breast cancers shows a transcriptional                        shift towards oxidative stress, DNA damage/repair, inflammation, hypoxia, and                        aerobic glycolysis. These findings are consistent with the "Reverse Warburg                        Effect" ,9. NotabThus,                        we avidly reviewed the literature regarding theories of neuronal functioning,                        neuronal stress, and neuro-degeneration, in the central nervous system and we                        stumbled upon the idea of "Neuron-Glia Metabolic Coupling" -18  In If                        these two processes are indeed analogous, then epithelial tumor cells have                        already learned to behave as neurons, using the stroma as a means of support                        and nourishment. Figure Myofibroblasts and                        mesenchymal stem cells are known to often express GFAP  -21, an iSlc1a2 and Slc1a3. Importantly, one of                        these two glial-specific glutamate transporters (Slc1a3) is also                        transcriptionally over-expressed in the stroma of human breast cancer patients                         from neurons. Then,                        astrocytes take up glutamate via high affinity sodium-dependent glutamate                        transporters, such as    Table . As suchVenn diagrams.                 In the Venn diagram of Figure                 (a)        the set of stromal genes that are                        upregulated in breast cancer tumor patients versus normal breast stroma; (b)        the set of stromal genes that are                        upregulated in recurrence positive versus recurrence negative breast cancer                        patients (c)        the set of stromal genes that are                        upregulated in lymph-node metastasis positive versus lymph-node metastasis                        negative breast cancer patients.                                In the Venn diagram of Figure Gene set enrichment analysis.                 For the functional analysis presented in Table                 For our analysis we used the latest release of MSigDB                        database v2.5 , after converting all the gene names in the                        database into RefSeq gene IDs. After this preprocessing step, we chose the                        sub-collection of gene sets that was relevant to our study, and for each gene                        set X in that sub-collection, we computed the overlap between X and the set of                        genes Y that are upregulated in lymph-node metastasis positive versus                        lymph-node metastasis negative breast cancer patients .                        Then, we computed the probability  of the observed overlap between                        sets X and Y as described in the "Venn diagrams" section.                                Supplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4-10


Human colorectal cancer tissue and matched uninvolved mucosa from 21 patients were examined by radioligand displacement for the presence of binding sites for bombesin-like peptides. Five cancers, but no uninvolved mucosa, expressed high-affinity, low-capacity bombesin binding sites  of the gastrin-releasing peptide (GRP)-preferring subtype (IC50 4.8 nM). Bombesin-like peptides may have a role in the pathogenesis of colorectal cancer, and bombesin receptor antagonists may be of value in the treatment of receptor-positive tumours.


Intrinsic low-level resistance to anti-cancer drugs is a major problem in the treatment of gastrointestinal malignancies. To address the problem presented by intrinsically resistant tumours, we have isolated two monoclonal lines from LoVo human colon adenocarcinoma cells: LoVo/C7, which is intrinsically resistant to doxorubicin (DOX); and LoVo/C5, which shows the same resistance index for DOX as the mixed parental cell population. For comparison, we have included in the study a LoVo-resistant line selected by continuous exposure to DOX and expressing a typical multidrug resistant (MDR) phenotype. In these cell lines we have studied the expression and/or activity of a number of proteins, including P-glycoprotein 170 (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione (GSH)-dependent enzymes and protein kinase C (PKC) isoforms, which have been implicated in anti-cancer drug resistance. Intracellular DOX distribution has been assessed by confocal microscopy. The results of the present study indicate that resistance in LoVo/C7 cells cannot be attributed to alterations in P-gp, LRP or GSH/GSH-dependent enzyme levels. Increased expression of MRP, accompanied by alterations in the subcellular distribution of DOX, has been observed in LoVo/C7 cells; changes in PKC isoform pattern have been detected in both intrinsically and pharmacologically resistant cells.


Recent studies identified an interaction between the Polymerase Associated Factor complex (PAFc) and Mixed Lineage Leukemia (MLL), including MLLrearranged oncoproteins. This interaction is critical for MLL transcriptional activity and MLL-rearranged leukemogenesis. Here, we discuss the potential molecular role of the PAFc in transcriptional dysregulation of MLL target genes and the interplay between PAFc and MLL-rearranged oncoproteins in leukemogenesis. MLL, the human homolog of the Drosophila trithorax gene, encodes a histone H3 lysine 4 (H3K4) methyltransferase that positively regulates multiple homeobox transcription factors, including Hoxa9 and MEIS1, which are pivotal for leukemogenesis [MLL rearrangements that generate MLL-rearranged oncoproteins are associated with a variety of acute lymphoid and myeloid leukemias that have a dismal prognosis [MLL-related translocations are also commonly observed in secondary acute leukemias after topoisomerase inhibitor treatment [MLL-PTD  and the SET domain, which is required for the histone methyltransferase activity, are consistently deleted  interacts with this region and that this interaction is critical for MLL transcriptional activity as well as leukemogenesis [Our recent study, as well as the work from Milne ogenesis , 12. PAFogenesis -16. Incrogenesis -19. In aogenesis . Previouogenesis , 20. Givogenesis .MLL-rearranged leukemogenesis. The best defined target genes of MLL are the clustered homeobox (Hox) genes, a transcription factor family important in cell fate determination during development. Among these targets, Hoxa9 and its cofactor Meis1 have been shown to be crucial for MLL-rearranged leukemogenesis. Normally, Hoxa9 and Meis1 are only briefly expressed in hematopoietic stem cell and progenitor cell populations and are then rapidly down regulated during hematopoietic differentiation [Characterization of the PAFc-MLL interaction provides valuable insight into the mechanisms of ntiation -23. HoweHox gene dysregulation in MLL-rearranged leukemias. First, increasing evidence suggests that a significant mechanism for Hox gene expression is mediated through regulating transcriptional elongation [Hoxa9 promoter induced by MLL-AF9 overexpression, whereas wild-type MLL overexpression only delivered a somewhat more muted response, suggesting the differential roles of PAFc in cellular activities of MLL-rearranged oncoproteins vs. wild-type MLL. Third, a previous study by Chen et al. has demonstrated that the susceptibility of hematopoietic progenitors to MLL-AF9-induced transformation decreases along differentiation [Hoxa9 and Meis1, to constitutively activate transcription, leading to leukemogenesis. Therefore, PAFc may be a crucial component mediating the dysregulation of normal transcription elongation of MLL target genes by MLL-rearranged oncoproteins  stem cells, but not the more differentiated committed granulocyte-monocyte progenitors (GMPs), can be transformed by MLL-AF9 under endogenous regulatory control, suggesting that under physiological conditions, additional linage-specific transcription factor(s) or coactivators(s), other than the MLL-rearranged oncoproteins are critical for leukemogenesis [The most common MLL-rearranged oncoproteins, including MLL-AF9, MLL-ENL and MLL-AF4, are known to interact with a protein complex termed ENL-associated proteins (EAP) or a closely related complex called AEP for AF4 family/ENL family/P-TEFb complex . By inteogenesis . Given tMLL translocations resulting in MLL fusion proteins with a nuclear translocation partner, MLL fusion proteins with cytoplasmic partners and MLL-PTD have not been extensively studied. Therefore, the leukemogenic mechanisms of MLL-PTD and MLL-rearranged oncoproteins with a cytoplasmic partner, both of which in effect involve duplication of the N-terminus of MLL (up to the breakpoint region) by either intramolecular partial tandem duplication or intermolecular dimerization, are unknown [A number of questions remain regarding the mechanism of the PAFc-MLL interaction in MLL-rearranged leukemogenesis. First, apart from the most common  unknown . Given t


Rk confers resistance against several species of root-knot nematodes  in cowpea (Vigna unguiculata). Based on histological and reactive oxygen species (ROS) profiles, Rk confers a delayed but strong resistance mechanism without a hypersensitive reaction-mediated cell death process, which allows nematode development but blocks reproduction.The locus M. incognita infection in roots of resistant genotype CB46 and a susceptible near-isogenic line (null-Rk) were investigated using a soybean Affymetrix GeneChip expression array at 3 and 9 days post-inoculation (dpi). At 9 dpi 552 genes were differentially expressed in incompatible interactions (infected resistant tissue compared with non-infected resistant tissue) and 1,060 genes were differentially expressed in compatible interactions (infected susceptible tissue compared with non-infected susceptible tissue). At 3 dpi the differentially expressed genes were 746 for the incompatible and 623 for the compatible interactions. When expression between infected resistant and susceptible genotypes was compared, 638 and 197 genes were differentially expressed at 9 and 3 dpi, respectively.Responses to In comparing the differentially expressed genes in response to nematode infection, a greater number and proportion of genes were down-regulated in the resistant than in the susceptible genotype, whereas more genes were up-regulated in the susceptible than in the resistant genotype. Gene ontology based functional categorization revealed that the typical defense response was partially suppressed in resistant roots, even at 9 dpi, allowing nematode juvenile development. Differences in ROS concentrations, induction of toxins and other defense related genes seem to play a role in this unique resistance mechanism. Vigna unguiculata L. Walp) is grown extensively as a food and fodder crop in West Africa, lower elevation areas of eastern and southern Africa, north-eastern Brazil, parts of the Middle East, India, and the south-eastern and south-western regions of North America )Principal component analysis (PCA) is often used to reduce multidimensional data sets to lower dimensions for summarizing the most important part of the data while simultaneously filtering out the background errors. PCA involves the calculation of the eigenvalue decomposition of a data covariance matrix or singular value decomposition of a data matrix, usually after mean centering the data for each attribute. The results of PCA are usually discussed in terms of component scores and loadings . PCA on For the 3-dpi samples, with only two biological replicates available, a Pearson correlation coefficient was calculated for normalized values of all probe sets between the two replicates of each treatment to determine the robustness of the data. This analysis was carried out in dChip software .Differentially expressed genes were identified using a one-way analysis of variance (ANOVA) with a p-value cut-off of 0.05. A multiple testing correction was performed using the Bonferroni error correction model . False dIn our recent related work  the AffyArabidopsis translated gene models (version 7.0) from The Arabidopsis Information Resource  and Medicago truncatula 2.0 assembly release http://www.medicago.org. Annotations for the Affymetrix soybean probe sets were compiled into a browser called HarvEST:SoyChip which can be accessed online http://www.harvest-web.org or downloaded for Windows installation http://harvest.ucr.edu/. The E value cut-off for the gene annotations was equal to or less than E-10, E0 being a near perfect match.The soybean genome array unigene sequences were used to query (using blastx) Arabidopsis gene models to Munich Information Center for Protein Sequences Arabidopsis thaliana FunCat database . Arabidopsis gene models were taken from HarvEST:SoyChip.Gene ontology based classification was obtained by transferring the corresponding SD, PAR and TJC designed the experiments. SD performed the research and did the data analysis. SD, PAR and TJC wrote the paper. JDE provided plant materials. All the authors have read and approved the final manuscript.Principal component analysis (PCA) plot of cowpea genome response to nematode infection at 3 days post-inoculation. Each dot represents the mean of a particular condition (treatment).Click here for fileGenes passing 1.5-fold filter in the incompatible cowpea-RKN interaction (9 days post-inoculation).Click here for fileGenes passing 1.5-fold filter in the compatible cowpea-RKN interaction (9 days post-inoculation).Click here for fileRk compared to the infected null-Rk (9 days post-inoculation)Genes passing 1.5-fold filter in infected .Click here for fileGenes passing 1.5-fold filter in the incompatible cowpea-RKN interaction (3 days post-inoculation).Click here for fileGenes passing 1.5-fold filter in the compatible cowpea-RKN interaction (3 days post-inoculation).Click here for fileRk compared to the infected null-Rk (3 days post-inoculation)Genes passing 1.5-fold filter in infected .Click here for fileGenes differentially expressed in both 9- and 3-dpi samples in cowpea-RKN incompatible interaction.Click here for fileGenes differentially expressed in both 9- and 3-dpi samples in cowpea-RKN compatible interaction.Click here for file


Post-mortem studies suggest that liver metastases obtain the majority of their nutrition from the hepatic artery; however, cytotoxic arterial perfusion with or without hepatic-artery ligation has not proved entirely successful as a therapeutic regime. In this study we have measured blood flow into colorectal liver metastases using xenon-133 (133Xe) clearance in patients undergoing surgery for colorectal cancer. Pre-operative measurements after direct parenchymal injection gave a mean flow of 41.5 +/- 22.5 ml/min/100 g which after hepatic arterial occlusion perfusion, was reduced to a mean of 5% of the pre-occlusion value. Dynamic blood-flow studies using the gamma camera were performed in the post-operative period by administration of 133Xe into both hepatic arterial and portal venous catheters. The initial distribution images indicated a predominant arterial perfusion to the metastases, but after hepatic-artery ligation, portal-vein perfusion to the metastases was statistically significantly increased. Hence, a compensatory haemodynamic mechanism exists which may account for the poor results of hepatic-artery ligation and perfusion alone.


The aim of this study was to investigate mechanisms of anti-tumour activity and necrosis induced by combinations of tumour necrosis factor alpha  and interferon gamma (IFN-gamma). In a breast cancer xenograft model, locally injected recombinant human TNF-alpha arrested growth of established tumours in the absence of overt necrosis. Macroscopic necrosis occurred when rat IFN-gamma, which had no anti-tumour activity as a single agent, was given systemically. Treatment with TNF-alpha and IFN-gamma caused focal engorgement of tumour capillaries with erythrocytes, intravascular recruitment of polymorphonuclear cells and platelet adherence to the tumour vascular endothelium 4 h after the combined treatment. This was followed by destruction of tumour vascular endothelium and both necrosis and apoptosis of tumour cells. Concomitant with these changes, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the increase of stromal (murine) mRNA levels for TNF-alpha, TNF receptor 55 kDa, TNF receptor 75 kDa, intracellular adhesion molecule 1, vascular cell adhesion molecule 1, P-selectin and interleukin 6 (IL-6). Thus, the effect of the combined TNF-alpha and IFN-gamma therapy involved the selective destruction of the tumour vasculature, death of tumour cells and increased expression of a series of stromal cytokines, cytokine receptors and adhesion molecules, which could be implicated in the observed events.


We have compared the actions of ADM and mAMSA in Chinese hamster V79 cells in vitro, using cell survival and sister-chromatid exchange as end-points. Equimolar concentrations of ADM and mAMSA show similar toxicities to exponentially growing cells, and both drugs are less effective in killing chronically hypoxic and plateau-phase cells. Cytotoxicity to thermotolerant cells (41 degrees C for 16 h previously) shows little difference from that for exponential cells. Pre-treating cells with misonidazole under hypoxic conditions reduces the toxicity of both ADM and mAMSA. In addition, an ADM-resistant Chinese hamster cell line, 77A-177, was cross-resistant to mAMSA. Finally, low equimolar sub-toxic doses of both drugs were found to cause similar increases in the levels of sister-chromatid exchanges in V79 cells. These results reveal no major difference in activity between ADM and mAMSA in vitro.


The cellular reservoirs of Kaposi's sarcoma-associated herpesvirus (KSHV) and the exact nature of the putative KSHV-infected circulating precursor of spindle cells of Kaposi's sarcoma (KS) still remain poorly defined. Because KS spindle cells are thought to be of endothelial origin, and because mature endothelial cells do not sustain persistent KSHV-infection, our attention was focalized on circulating hematopoietic precursors able to differentiate into endothelial lineage.Late endothelial progenitor cells (late-EPCs) were cultured from the peripheral blood mononuclear cells of 16 patients with classic KS. The presence and load of KSHV genomes were analyzed by real-time polymerase chain reaction in DNA extracted from cells and supernatants of late-EPC cultures obtained from 7 patients. Endothelial colonies cultured from the peripheral blood of KS patients were found to satisfy all requisites to be defined late-EPCs: they appeared from the CD14-negative fraction of adherent cells after 11–26 days of culture, could be serially expanded in vitro, expressed high levels of endothelial antigens but lacked leukocyte markers. Late-EPC cultures were found to harbor KSHV-DNA at variable levels and to retain the virus after multiple passages in cells as well as in supernatants, suggesting that a quote of KSHV lytic infection may spontaneously occur. Lytic phase induction or hypoxia could amplify virus release in supernatants.Our results suggest that circulating endothelial progenitors from KS patients are KSHV-infected and support viral productive replication and may therefore represent potential virus reservoirs and putative precursors of KS spindle cells. Kaposi's sarcoma (KS) is a multifocal angioproliferative disease of the skin and mucosa. Central to its pathogenesis is a hyperproliferation of spindle-shaped cells, which are the prominent and hallmark cells of KS lesions. Spindle cells are thought to be of endothelial origin that assume the characteristic spindle shape upon infection with KS-associated herpesvirus (KSHV), also called human herpesvirus-8 (HHV-8), the causative agent for KS In KS patients multiple lesions, supposed to be multiclonal in origin A clear-cut identification of putative KSHV-infected endothelial progenitors in the peripheral blood of KS patients is still lacking. Therefore, in the present study we cultured circulating endothelial progenitor cells from the blood of patients with classic KS (cKS) and assessed their status of KSHV infection.Sixteen patients with cKS were included in the study, 10 males and 6 females, mean age 70 years (range 47–82). All patients had histologically confirmed diagnosis of KS, were positive for anti-HHV8 antibodies and negative for HIV. Patients in systemic chemotherapy were excluded. Staging was performed in accordance with our classification that takes into account the prevalent type of lesions, localization, clinical behaviour, evolutive pattern and presence of complications 6 cells/well) precoated with human fibronectin . Four to eight wells (mean 6.07±s.e. 0.37) were plated for each patient. In two experiments, PBMCs underwent separation of CD14+ and CD14- fractions using immunomagnetic selection with mini-magnetic-activated cell sorter (MACS) cell isolation kit  according to the manufacturer's instructions. After 3 days of culture, nonadherent cells and debris were aspirated, adherent cells were washed with EGM-2 medium, and EGM-2 medium was added to each well. Medium was changed on day 7 and every 3 days until the first passage. Late-EPC colonies were identified by visual inspection using an inverted microscope. Late-EPCs were released from the original tissue culture plates by trypsinization (trypsin 0.25%) , resuspended in EGM-2 medium, and plated onto 25 cm2 tissue culture flasks precoated with fibronectin for further passages. Multiple colonies from the same patient were pooled at this step. In one single experiment, late-EPCs obtained from a KS patient underwent cell sorting of CD146+ cells . Purity of CD146+ cells before and after sorting was 98.7% and 100%, respectively. This highly pure population of CD146+ late-EPCs was replated and maintained in culture for further two weeks for virus analyses.Late-EPCs were generated from peripheral blood mononuclear cells (PBMCs) of cKS patients according to methods described by Ingram Cells were cultured onto fibronectin-coated, 2-chamber Lab-Tek slides  and attached cells were incubated with 10 µg/ml Dil-acetylated-low-density lipoprotein (Dil-ac-LDL)  in EGM-2 medium for 1 hour at 37°C. Cells were then fixed with 2% formaldehyde for 10 minutes and incubated for 1 hour with fluorescein-isothiocyanate (FITC)-labeled Ulex Europaeus Agglutinin-1 . Alternatively, cells were fixed with methanol, permeabilized with 0.1% Triton X-100, blocked with 10% normal goat serum  and incubated with purified antibodies against human von Willebrand Factor (vWF), endothelial nitric oxide synthase (e-NOS) or caveolin-1 (Cav-1) , followed by appropriate FITC- or phycoerythrin (PE)-conjugated secondary antibodies . Slides were mounted in anti-fading mounting media containing DAPI  and examined by conventional fluorescence-microscopy .5 late-EPCs were incubated with different anti-human monoclonal antibodies (mAbs) for 30 minutes at 4°C in the dark. The following FITC-, PE-, allophycocyanin (APC)- or biotin-conjugated mAbs were used: anti-CD31, -CD45, (Becton-Dickinson); -kinase insert domain receptor (KDR) (Sigma-Aldrich), -CD105 (Euroclone), -CD14 , -CD146 , -CD34 (Miltenyi Biotec). The FASER technique was performed for CD34 detection according to manufacturer's instructions . Isotype-matched irrelevant mAbs were used as negative controls. Data were acquired on a FACSCanto flow cytometer (Becton-Dickinson) and analyzed using FACSDiva software. Cells were electronically gated according to light scatter properties to exclude cell debris.For antigen detection by flow cytometry, 5×104 cells per well, and incubated at 37°C. Cells were observed during the following 12–72 hours with inverted microscopy for capillary-like formation.Matrigel assays were performed as described 5 cells and from 200 ul of DNAse-treated culture supernatants as previously described n-butyrate (Sigma-Aldrich), or with 20 ng/ml of phorbol 12-myristate 13-acetate  Plasma samples were analyzed for the presence and titers of antibodies to a latency-associated nuclear antigen (LANA) and to a lytic phase-associated structural protein encoded by ORF65, as previously described in vitro induction of KSHV lytic replication. Analysis was performed with OPENSTAT3 software.The Wilcoxon signed-rank test was used to analyze the statistical significance of data obtained in the 6 PBMCs was 0.090±0.020, equivalent to 2.81±0.39 colonies from each patient. Late-EPCs proliferated slowly during the first weeks, but then proliferated more rapidly and could be serially expanded in vitro through multiple passages, up to 7–10 weeks, when they finally stopped growing and entered senescence. During these passages the cells retained their endothelial morphology, forming cobblestone-like monolayers . Also the amounts of viral genomes spontaneously released by late-EPCs in the supernatants were similar before and after sorting , thus suggesting that the negligible proportion of CD146-negative cells did not represent the source of virus in our cultures.KSHV DNA presence and load was determined by real-time PCR in PBMCs and late-EPC cultures from 7 cKS patients, and in one KSHV-seronegative healthy control, as shown in n-butyrate or TPA, whereas cultures from three other patients had sufficient cells to be treated only with n-butyrate. KSHV genomes were measured by real-time PCR in culture supernatants 48 hours after induction. As reported in P = 0.009), suggesting that late-EPCs may support KSHV productive replication. As shown in n-butyrate treatment of sorted CD146+ late-EPCs (from patient KS 7) increased the release of KSHV genomes in the supernatants to the same extent as unsorted cells , thus suggesting that late-EPCs with proven endothelial phenotype can support viral lytic induction.To induce the viral lytic cycle in KSHV-infected late-EPCs, cultures from two patients were treated in parallel with The effects of late-EPC exposure to prolonged hypoxia, which may act in vivo as a cofactor in the development of KS lesions, were evaluated in 2 patients. In both cultures, hypoxia induced an increase in KSHV levels in culture supernatants. In facts, late-EPCs from patient KS 5 showed a 5-fold increase in virus release (from 906 to 4454 GE/ml), whereas a moderate increment was observed in the culture of patient KS 6 (from 564 to 690 GE/ml). Late-EPCs obtained from the healthy negative control were found to be repeatedly negative for KSHV DNA sequences also in hypoxic culture conditions (not shown).In this report, we provide evidence that late-EPCs from the peripheral blood of patients with cKS harbor KSHV, may support lytic replication and may act, therefore, as viral reservoirs. The novelty of this finding resides in the nature of late-EPCs. Among other cell populations with endothelial features circulating in adult peripheral blood, late-EPCs, also called outgrowth endothelial cells (OECs) First of all, in this study we demonstrate that late-EPCs can be cultured from the peripheral blood of patients with cKS. Moreover, our results clearly indicate that late-EPCs from cKS are infected by KSHV and may retain the virus in long-term cultures, as late-EPCs from the totality of cKS patients studied resulted to harbor variable amounts of viral genomes in cultures tested up to over 2 months.Infected spindle cells explanted from KS biopsies are known to rapidly segregate and loose KSHV genomes when placed in culture The detection of viral progeny released in all the supernatants of unstimulated cultures indicates that late-EPCs may support the lytic replication program of KSHV and this finding may alternatively suggest that the virus is propagated in culture by cycles of de novo infection of late-EPCs. Accordingly, when KSHV DNA was analyzed in subsequent passages of unstimulated late-EPCs from patient KS 4, we could observe that the levels of viral genomes may change over time. Whether these fluctuations may reflect cycles of productive viral replication that may be responsible for the continuous infection of endothelial progenitors may deserve further investigation.Moreover, our results indicate that the basal level of lytic replication can be further increased not only by chemical treatment but also by hypoxic conditions. This finding is worthy relevant to the role that lytic KSHV replication is supposed to play in the promotion of KS tumorigenesis by sustaining the population of latently infected cells In addition, the fact that late-EPCs harbor moderate amounts of viral genomes in unstimulated conditions may be partly related to the type of KS patients selected for the study. In fact, to avoid the interference of confounding factors related to iatrogenic or AIDS-related KS, we studied patients affected by the classic, Mediterranean variant of the disease that is typically characterized by a poorly aggressive course and a relatively low viral load Endothelial progenitors are thought to be contained, together with hematopoietic stem/progenitor populations, within the pool of circulating CD34+ cells In conclusion, in this study we suggest that circulating endothelial progenitor cells cultured from the peripheral blood of cKS patients as late-EPCs are infected by KSHV and can support viral lytic replication. This finding may be of crucial importance for the comprehension of KS pathogenesis, because late-EPCs may likely represent the KSHV-infected circulating precursors of KS spindle cells that, as recently suggested by Gill, may home to the permissive site and propagate to produce KS lesions


Simultaneous spectrophotometric determination of Mn, Zn and Co was studied by two methods, classical partial least-squares (PLS) and kernel partial least-squares (KPLS), with 2-(5-bromo-2- pyridylazo)-5-diethylaminephenol (5-Br-PADAP) and cetyl pyridinium bromide (CPB). Two programs, SPGRPLS andSPGRKPLS, were designed to perform the calculations. Eight error functions were calculated for deducing the number of factors. Data reductions were performed using principle component analysis. The KPLS method was applied for the rapid determination from a data matrix with many wavelengths and fewer numbers of samples. The relative standard errors of prediction (RSEP) for all components with KPLS and PLS methods were the same(0.0247). Experimental results showed both methods to be successful even where there was severe overlap of spectra.


Exaggerated production of superoxide and inactivation of nitric oxide have been implicated in pathogenesis of hypertension. NAD(P)H oxidase is one of the major source of reactive oxygen species in vasculature. In the present study, we aimed to determine the effect of chronic administration of Apocynin an NAD(P)H oxidase inhibitor on endothelial function and hypertension in fructose-fed rat.Endothelial function, vascular superoxide, and nitric oxide production/bioavailability in aortas from fructose-fed rats and age-matched controls treated with or without apocynin were assessed using isometric tension studies in organ chambers. Systolic blood pressure was measured by the tail cuff method.In fructose-fed rats, acetylcholine-induced relaxation was impaired, vascular superoxide production was increased, and nitric oxide bioavailability was decreased along with an increase in systolic blood pressure compared to controls. Apocynin treatment prevented the increased generation of superoxide, decreased nitric oxide bioavailability, impaired acetylcholine-induced relaxation, and elevation of systolic blood pressure.Chronic administration of apocynin improves the endothelial function by reducing oxidative stress, improving NO bioavailability, and prevents the development hypertension in fructose-fed rat. Note, this impaired response to Ach was normalized by treatment with the NAD(P)H oxidase inhibitor apocynin  [2 values among the groups. The sensitivity (pD2) and maximum relaxation response (Emax) induced by the endothelium-independent vasodilator sodium nitroprusside were not significantly different among the groups [The endothelium-dependent vasodilator acetylcholine (Ach) and endothelium-independent vasodilator (SNP) elicited the concentration-dependent relaxation of phenylephrine preconstricted isolated aortic rings of all animals Figures  and 3. A < 0.05) . The sene groups .2− in aorta isolated from fructose-fed rats compared with levels in aorta isolated from control. This increase was prevented in aortic rings isolated from apocynin-treated fructose-fed rats [2− level.A significant increase in tempol (SOD mimetic)-induced relaxation of isolated rat aorta preconstricted with phenylephrine revealed a marked increase in vascular Ofed rats . HoweverA significant decrease in L-NAME-induced contraction of isolated rat aorta preconstricted with phenylephrine revealed a marked decrease in vascular NO bioavailability in aorta isolated from fructose-fed rats compared with control. This decrease was prevented in apocynin-treated fructose-fed rat . HoweverIn the present study, we demonstrate for the first time that chronic administration of apocynin, a putative NAD(P)H oxidase inhibitor, prevents the development of endothelial dysfunction and hypertension by inhibiting exaggerated vascular superoxide production and increasing the bioavailability of nitric oxide, supporting the hypothesis that reduced NO bioavailability due to increased reactive oxygen species  plays a critical role in the development of endothelial dysfunction and hypertension in fructose-fed rat.2− level is known to inactivate the vasodilator nitric oxide, leading to endothelial dysfunction, a characteristic feature of many vascular diseases, including hypertension.[Growing body of evidence supports the role of ROS in pathogenesis of hypertension. This notion is supported by the observation that vascular ROS production is elevated in different experimental models of hypertension.–9 Increartension.–17 In vartension.–20 In thrtension.22Picrorhiza kurroa, a orally active reversible inhibitor of NAD(P)H oxidase, has been reported to inhibit the activation of NAD(P)H oxidase by blocking the assembly of a functional NADPH oxidase complex.[2− production and attenuates hypertension in hypertensive rats.[Apocynin, a methoxy-substituted catechol from the herb  complex. Administive rats.–26 Thus,ive rats.–27in vitro incubation with apocynin prevents this increase in vascular superoxide production.[9Fructose-fed rat, a widely used animal model of insulin resistance syndrome, exhibits oxidative stress, endothelial dysfunction, and hypertension. Treatment with antioxidants has been shown to reduce blood pressure in this model.29 Recentduction.9 However,duction.931 Thus, in vitro incubation with apocynin prevents this increase in vascular superoxide production.[9Fructose-fed rat, a widely used animal model of insulin resistance syndrome, exhibits oxidative stress, endothelial dysfunction, and hypertension. Treatment with antioxidants has been shown to reduce blood pressure in this model.29 Recentduction.9 However,duction.931 Thus, This work demonstrates that NAD(P) H oxidase-mediated excessive superoxide production may reduce the bioavailability of NO which may account for endothelial dysfunction and hypertension in fructose-fed rats. These findings may provide additional evidence/basis for therapeutic interventions aimed at reducing superoxide-induced vascular dysfunctions associated with insulin resistance and hypertension.


Alteration in MHC class I expression may be used by cancer cells to avoid immune destruction. Much experimental evidence supports this idea, although survival studies are very scarce. To investigate whether the presence or absence of HLA-A, -B and -C antigens in laryngeal carcinoma influences survival, a series of 60 primary laryngeal tumours treated surgically and normal tissues were evaluated in frozen sections for the expression of MHC class I antigens and tumour-infiltrating leucocytes , using monoclonal antibodies and the APAAP, technique. Long-term follow-up from the patients is available, ranging from 6 to 10 years. Thirteen tumours presented total HLA-ABC loss, five selective losses of HLA-A antigens and one absence of HLA-B antigens. Total losses were statistically associated with several clinical and pathological parameters, but there were no differences regarding tumour-infiltrating leucocytes. After conducting a prospective study, only T and N staging and scoring according to Glanz's malignancy classification were found to be independently related to patients' outcome. From our data, we conclude that neither complete loss of HLA class I antigens nor tumour-infiltrating leucocytes appear to influence survival in squamous cell carcinoma of the larynx.


Magnolia officinalis, were investigated on animal paw licking responses and thermal hyperalgesia induced by glutamate receptor agonists including glutamate, N-methyl-D-aspartate (NMDA), and metabotropic glutamate 5 receptor (mGluR5) activator (RS)-2-chloro-5-hydroxyphenylglycine (CHPG), as well as inflammatory mediators such as substance P and prostaglandin E2 (PGE2) in mice. The actions of honokiol and magnolol on glutamate-induced c-Fos expression in the spinal cord dorsal horn were also examined. Our data showed that honokiol and magnolol blocked glutamate-, substance P- and PGE2-induced inflammatory pain with similar potency and efficacy. Consistently, honokiol and magnolol significantly decreased glutamate-induced c-Fos protein expression in superficial (I-II) laminae of the L4-L5 lumbar dorsal horn. However, honokiol was more selective than magnolol for inhibition of NMDA-induced licking behavioral and thermal hyperalgesia. In contrast, magnolol was more potent to block CHPG-mediated thermal hyperalgesia. These results demonstrate that honokiol and magnolol effectively decreased the inflammatory pain. Furthermore, their different potency on inhibition of nociception provoked by NMDA receptor and mGluR5 activation should be considered.The antinociceptive effects of honokiol and magnolol, two major bioactive constituents of the bark of  Magnolia officinals  induced by intraplantar injection of PGEThe highest counts of c-Fos-positive cells were observed at superficial (I-II) laminae of the L4-L5 lumbar dorsal horn ipsilateral to the glutamate-injected site Fig. . In cont2-induced nociceptive responses. The inhibitory actions of honokiol on NMDA-induced licking behavioral and thermal hyperalgesia were more potent than magnolol. In contrast, magnolol had higher potency than honokiol to block CHPG-mediated thermal hyperalgesia. Importantly, both honokiol and magnolol significantly decreased glutamate-induced c-Fos protein expression in superficial (I-II) laminae of the L4-L5 lumbar dorsal horn. Taken together, these results indicated that the analgesic actions of honokiol and magnolol may be related to the blockade of glutamate receptor, NMDA receptor and mGlu5 receptor activation, and inflammation-mediated nociception.Our previous report has indicated that honokiol and magnolol possess the ability to inhibit formalin-induced inflammatory nociception . The preAccumulating evidence has demonstrated that the excitatory amino acid glutamate and the glutamatergic receptors, both ionotropic and metabotropic glutamate receptors (iGluR and mGluRs), are critically contributed to nociceptive neurotransmission under the development and maintenance of pain responsiveness -19. In r2+]i or neurotoxicity in cerebellar granule cells [It has been demonstrated that noxious stimuli-induced release of glutamate from nociceptive afferents in spinal cord, leading to the activation of either iGluR or mGluR on the postsynapse . Fos, a le cells ,11. In c2 is the prostanoids most relevant for the induction and maintenance of the inflammatory pain by either increasing the primary nociceptor responses or changing the spinal nociceptive signaling [2 are attributed to the activation of G-protein-coupled EP receptors located in intrinsic spinal neurons (EP2) or primary afferent neurons  and protein kinase dependent mechanisms [2+ and Na+ conductance [2-induced hyperalgesia is abolished in NMDA receptor knock-out mice [2 but also blocked these responses induced by NMDA and glutamate. Thus, the blockade of glutamate receptors by honokiol and magnolol could contribute to their antinociception on PGE2-induced pain responses. However, the antinociceptive principles for interfering at the EP receptors or the following signaling transduction in PGE2-induced thermal hyperalgesia can not be excluded. Further study is required to figure out the analgesic mechanisms of honokiol and magnolol on PGE2-mediated nociceptive and inflammatory responses.During an inflammatory event, nociception generation is a consequence of a complex interaction between a number of inflammatory mediators, including prostaglandins (PG), in inflamed tissues and the spinal cord. Notably, PGEignaling . The nocchanisms ,32. Prosductance . Furtherout mice . The preUpon noxious stimulation, tachykinins such as substance P and neurokinin A that are released from primary afferent fibers and excite spinal cord dorsal horn neurons bind to neurokinin receptors. It is believed that substance P induced signaling pathway is involved in the inflammatory process and in the transmission of sensory nociceptive information ,35,36. A2- and substance P-induced pain, and consistently decrease glutamate-induced c-Fos protein expression in superficial (I-II) laminae of the L4-L5 lumbar dorsal horn. It is noted that honokiol preferentially modulates the NMDA receptor-mediated nociception, whereas magnolol is more potent than honokiol to inhibit mGluR5-mediated response. Therefore, these results indicate that honokiol and magnolol may possess the therapeutic potential to treat the inflammatory pain and have the differential analgesic mechanisms on NMDA receptor and mGluR5 activation under nociceptive neurotransmission.In conclusion, the present study demonstrates that honokiol and magnolol produce the antinociception against glutamate-, NMDA-, CHPG-, PGEThe authors declare that they have no competing interests.YRL carried out the nociceptive tests and immunohistochemical assay, measured the thermal hyperalgesia, and drafted the manuscript. HHC participated in the design of the study and sequence alignment. YCL and CHK participated in the coordination and sequence alignment. MHC conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Theglycolipid sulphatide was shown to block the adherence ofthrombin-activated rat platelets to HL-60 cells. This interaction isknown to be dependent on P-selectin. The rat dermal reverse passiveArthus reaction was used to assess the effect of sulphatide on aneutrophil dependent inflammatory response. Sulphatidedosedependently blocked both the vascular permeability increase andcell infiltration after intraperitoneal administration. Theseresults show that a small molecular weight compound which blocks P-and E-selectin dependent adhesion


Within these PBMCs, circulating             B cells were differentiated in vitro and produced IgG Abs against not only ENV, but             also GAG and POL proteins. Under similar experimental conditions, HAART treated              patients produced Abs to ENV proteins only. The in vitro antibody production by              drug-naïve individuals' PBMCs depended on exogenous cytokines (IL-2 and IL-10)               but neither on the re-stimulation of reactive cells in cultures by purified HIV-1-gp               160 antigen nor on the re-engagement of CD40 surface molecules. Further, it was                not abrogated by the addition of various monoclonal Abs (mAbs) to co-stimulatory                 molecules. This suggests that the in vitro antibody production by drug-naïve                 individuals' PBMCs resulted from the maturation of already envelope and core                  antigen-primed, differentiated B cells, presumably pre-plasma cells, which are                  not known to circulate at homeostasy. As in vitro produced Abs retained the                  capacity of binding antigen and forming complexes, this study provides                  pre-clinical support for functional humoral            responses despite major HIV- and other tropical pathogen-induced B cell            perturbations.Unseparated peripheral blood mononuclear cells (PBMCs) obtained from            drug-naïve African individuals living in a context of multi-infections and presenting             with high viral load (VL), were cultured