Acute toxicity of a single gavage dose of fumonisin B(1) in rabbits

The aim of this study was to determine the clinical, pathological and mycotoxicological effects of oral administration of fumonisin B, (FBI) in rabbits. Eighteen rabbits were randomly assigned to two experimental groups: control group, 0 mg FB(1): fumonisin group. 31.5 mg FB(1)/kg body weight, corresponding to about 630 mg FB(1)/kg diet. Fumonisin administered as a single oral dose to rabbits resulted in acute toxicity, significantly interfering with body and liver weight. Serum biochemical analysis revealed a significant increase of total protein, alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), urea and creatinine in the group receiving FBI compared to control animals, a finding characterizing hepatic and renal injury in this group. Urinary protein concentrations were markedly elevated at 12,24,48 and 72 h after dosing, although visible pathological abnormalities were not observed, probably because of rapid repair of the damage. FBI was detected in feces, with a maximum concentration at 24h after administration, indicating that the enterohepatic circulation is important in rabbits. FBI concentrations found in urine were low, with peak elimination at 12 h after intoxication. The highest FBI concentrations were observed in feces compared to urine and liver, demonstrating that feces are the main routes of excretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Neurodegeneration and Increased Production of Nitrotyrosine, Nitric Oxide Synthase, IFN-gamma and S100 beta Protein in the Spinal Cord of IL-12p40-Deficient Mice Infected with Trypanosoma cruzi

Background/Aim: Chagas` disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100 beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100 beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100 beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p<0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p<0.01) and NOS (544%, p<0.001). Moreover, the intensity of nitrotyrosine (16%, p<0.01), NOS (38%, p<0.05) and S100 beta (21%, p<0.001) immunoreactivities were also augmented. No IFN-gamma labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled.Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100 beta may contribute to NOS activation in the absence of IL-12. Copyright (C) 2009 S. Karger AG, Basel

PAS-1, a protein from Ascaris suum, modulates allergic inflammation via IL-10 and IFN-gamma, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effector phases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolated from Ascaris strum worms, has an inhibitory effect on lung allergic inflammation due to its ability to down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here, we investigated the role of IL-12, IFN-gamma and IL-10 in the PAS-1-induced inhibitory mechanism using a murine model of asthma. Wild type C57BL/6, IL-12(-/-), IFN-gamma(-/-) and IL-10(-/-) mice were immunized with PAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-I was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophil peroxidase activity in OVA + PAS-1-immunized wild type mice. This effect well correlated with a significant reduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1 antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-I was also observed in IL-12(-/-) mice, but not in IFN-gamma(-/-) and IL-10(-/-) animals. These data show that IFN-gamma and IL-10, but not IL-12, play an important role in the PAS-1 modulatory effect. (C) 2008 Elsevier Ltd. All rights reserved.

Gamma-linolenic acid inhibits both tumour cell cycle progression and angiogenesis in the orthotopic C6 glioma model through changes in VEGF, Flt1, ERK1/2, MMP2, cyclin D1, pRb, p53 and p27 protein expression

Background: Gamma-linolenic acid is a known inhibitor of tumour cell proliferation and migration in both in vitro and in vivo conditions. The aim of the present study was to determine the mechanisms by which gamma-linolenic acid (GLA) osmotic pump infusion alters glioma cell proliferation, and whether it affects cell cycle control and angiogenesis in the C6 glioma in vivo. Methods: Established C6 rat gliomas were treated for 14 days with 5 mM GLA in CSF or CSF alone. Tumour size was estimated, microvessel density (MVD) counted and protein and mRNA expression measured by immunohistochemistry, western blotting and RT-PCR. Results: GLA caused a significant decrease in tumour size (75 +/- 8.8%) and reduced MVD by 44 +/- 5.4%. These changes were associated with reduced expression of vascular endothelial growth factor (VEGF) (71 +/- 16%) and the VEGF receptor Flt1 (57 +/- 5.8%) but not Flk1. Expression of ERK1/2 was also reduced by 27 +/- 7.7% and 31 +/- 8.7% respectively. mRNA expression of matrix metalloproteinase-2 (MMP2) was reduced by 35 +/- 6.8% and zymography showed MMP2 proteolytic activity was reduced by 32 +/- 8.5%. GLA altered the expression of several proteins involved in cell cycle control. pRb protein expression was decreased (62 +/- 18%) while E2F1 remained unchanged. Cyclin D1 protein expression was increased by 42 +/- 12% in the presence of GLA. The cyclin dependent kinase inhibitors p21 and p27 responded differently to GLA, p27 expression was increased (27 +/- 7.3%) while p21 remained unchanged. The expression of p53 was increased (44 +/- 16%) by GLA. Finally, the BrdU incorporation studies found a significant inhibition (32 +/- 11%) of BrdU incorporation into the tumour in vivo. Conclusion: Overall the findings reported in the present study lend further support to the potential of GLA as an inhibitor of glioma cell proliferation in vivo and show it has direct effects upon cell cycle control and angiogenesis. These effects involve changes in protein expression of VEGF, Flt1, ERK1, ERK2, MMP2, Cyclin D1, pRb, p53 and p27. Combination therapy using drugs with other, complementary targets and GLA could lead to gains in treatment efficacy in this notoriously difficult to treat tumour.

Metabotropic glutamate receptors transduce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain

The prion protein (PrP(C)) is highly expressed in the nervous system, and its abnormal conformer is associated with prion diseases. PrP(C) is anchored to cell membranes by glycosylphosphatidylinositol, and transmembrane proteins are likely required for PrP(C)-mediated intracellular signaling. Binding of laminin (Ln) to PrP(C) modulates neuronal plasticity and memory. We addressed signaling pathways triggered by PrP(C)-Ln interaction in order to identify transmembrane proteins involved in the transduction of PrP(C)-Ln signals. The Ln gamma 1-chain peptide, which contains the Ln binding site for PrP(C), induced neuritogenesis through activation of phospholipase C (PLC), Ca(2+) mobilization from intracellular stores, and protein kinase C and extracellular signal-regulated kinase (ERK1/2) activation in primary cultures of neurons from wild-type, but not PrP(C)-null mice. Phage display, coimmunoprecipitation, and colocalization experiments showed that group I metabotropic glutamate receptors (mGluR1/5) associate with PrP(C). Expression of either mGluR1 or mGluR5 in HEK293 cells reconstituted the signaling pathways mediated by PrP(C)-Ln gamma 1 peptide interaction. Specific inhibitors of these receptors impaired PrP(C)-Ln gamma 1 peptide-induced signaling and neuritogenesis. These data show that group I mGluRs are involved in the transduction of cellular signals triggered by PrP(C)-Ln, and they support the notion that PrP(C) participates in the assembly of multiprotein complexes with physiological functions on neurons.-Beraldo, F. H., Arantes, C. P., Santos, T. G., Machado, C. F., Roffe, M., Hajj, G. N., Lee, K. S., Magalhaes, A. C., Caetano, F. A., Mancini, G. L., Lopes, M. H., Americo, T. A., Magdesian, M. H., Ferguson, S. S. G., Linden, R., Prado, M. A. M., Martins, V. R. Metabotropic glutamate receptors trans-duce signals for neurite outgrowth after binding of the prion protein to laminin gamma 1 chain. FASEB J. 25, 265-279 (2011). www.fasebj.org

Effects of glutamine supplementation on kidney of diabetic rat

Glutamine is the most important donor of NH(3) in kidney playing an important role in acid-base buffering system. Besides this effect, glutamine presents many other relevant functions in the whole body, such as a precursor of arginine in adult and neonates. In addition to these effects, some studies have shown that glutamine can potentiate renal disease. In the present study, the effect of short-term treatment (15 days) with glutamine on control and diabetic rats was investigated. Using biochemical, histological and molecular biology analysis from control and diabetic rats we verified that glutamine supplementation increase in pro-inflammatory interleukins (IL)-1 beta and IL-6 content in renal cortex and induce alteration in glomerular characteristics. This study showed that short-term treatment with glutamine in association with increased glucose levels could cause important alterations in glomerular morphology that may result in fast progression of kidney failure.

Morphine peripheral analgesia depends on activation of the PI3K gamma/AKT/nNOS/NO/K(ATP) signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3K gamma/AKT protein kinase B (AKT) and culminated in increasedactivation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Impaired Vasodilator Activity in Deoxycorticosterone Acetate-Salt Hypertension Is Associated With Increased Protein O-GlcNAcylation

Hyperglycemia, which increases O-linked beta-N-acetylglucosamine (O-GlcNAc) proteins, leads to changes in vascular reactivity. Because vascular dysfunction is a key feature of arterial hypertension, we hypothesized that vessels from deoxycorticosterone acetate and salt (DOCA-salt) rats exhibit increased O-GlcNAc proteins, which is associated with increased reactivity to constrictor stimuli. Aortas from DOCA rats exhibited increased contraction to phenylephrine (E(max) [mN] = 17.6 +/- 4 versus 10.7 +/- 2 control; n = 6) and decreased relaxation to acetylcholine (47.6 +/- 6% versus 73.2 +/- 10% control; n = 8) versus arteries from uninephrectomized rats. O- GlcNAc protein content was increased in aortas from DOCA rats (arbitrary units = 3.8 +/- 0.3 versus 2.3 +/- 0.3 control; n = 5). PugNAc (O- GlcNAcase inhibitor; 100 mu mol/L; 24 hours) increased vascular O- GlcNAc proteins, augmented phenylephrine vascular reactivity (18.2 +/- 2 versus 10.7 +/- 3 vehicle; n = 6), and decreased acetylcholine dilation in uninephrectomized (41.4 +/- 6 versus 73.2 +/- 3 vehicle; n = 6) but not in DOCA rats (phenylephrine, 16.5 +/- 3 versus 18.6 +/- 3 vehicle, n = 6; acetylcholine, 44.7 +/- 8 versus 47.6 +/- 7 vehicle, n = 6). PugNAc did not change total vascular endothelial nitric oxide synthase levels, but reduced endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) phosphorylation (P < 0.05). Aortas from DOCA rats also exhibited decreased levels of endothelial nitric oxide synthase(Ser-1177) and Akt(Ser-473) (P < 0.05) but no changes in total endothelial nitric oxide synthase or Akt. Vascular O-GlcNAc-modified endothelial nitric oxide synthase was increased in DOCA rats. Blood glucose was similar in DOCA and uninephrectomized rats. Expression of O- GlcNAc transferase, glutamine: fructose-6-phosphate amidotransferase, and O- GlcNAcase, enzymes that directly modulate O-GlcNAcylation, was decreased in arteries from DOCA rats (P < 0.05). This is the first study showing that O-GlcNAcylation modulates vascular reactivity in normoglycemic conditions and that vascular O- GlcNAc proteins are increased in DOCA-salt hypertension. Modulation of increased vascular O-GlcNAcylation may represent a novel therapeutic approach in mineralocorticoid hypertension. (Hypertension. 2009; 53: 166-174.)

Differential kinase requirement for enhancement of Fc gamma R-mediated phagocytosis in alveolar macrophages by leukotriene B(4) vs. D(4)

In alveolar macrophages, leukotriene (IT) B(4) and cysteinyl LTs (LTC(4), LTD(4) and LTE(4)) both enhance Fc gamma receptor (Fc gamma R)-mediated phagocytosis. In the present study we investigated the role of specific PKC isoforms (PKC-alpha and -delta), the MAP kinases p38 and ERK 1/2, and PI3K in mediating the potentiation of Fc gamma R-mediated phagocytosis induced by addition of leukotrienes to the AMs. It was found that exogenously added LTB(4) and LTD(4) both enhanced PKC-delta and -alpha phosphorylation during Fc gamma R engagement. Studies with isoform-selective inhibitors indicated that exogenous LTB(4) effects were dependent on both PKC-alpha and -delta, while LTD(4) effects were exclusively due to PKC-delta activation. Although both exogenous LTB(4) and LTD(4) enhanced p38 and ERK 1/2 activation, LTB(4) required only ERK 1/2, while LTD(4) required only p38 activation. Activation by both LTs was dependent on PI3K activation. Effects of endogenous LTs on kinase activation were also investigated using selective LT receptor antagonists. Endogenous LTB(4) contributed to Fc gamma R-mediated activation of PKC-alpha, ERK 1/2 and PI3K, while endogenous cysLTs contributes to activation of PKC-delta, p38 and PI3K. Taken together, our data show that the capacities of LTB(4) and LTD(4) to enhance Fc gamma R-mediated phagocytosis reflect their differential activation of specific kinase programs. (C) 2008 Elsevier Ltd. All rights reserved.

New malaria vaccine candidates based on the Plasmodium vivax Merozoite Surface Protein-1 and the TLR-5 agonist Salmonella Typhimurium FliC flagellin

The present study evaluated the immunogenicity of new malaria vaccine formulations based on the 19 kDa C-terminal fragment of Plasmodium vivax Merozoite Surface Protein-1 (MSP1(19)) and the Salmonella enterica serovar Typhimurium flagellin (FIiC), a Toll-like receptor 5 (TLR5) agonist. FHC was used as an adjuvant either admixed or genetically linked to the P. vivax MSP1(19) and administered to C57BL/6 mice via parenteral (s.c.) or mucosal (i.n.) routes. The recombinant fusion protein preserved MSP1(19) epitopes recognized by Sera collected from P. vivax infected humans and TLR5 agonist activity. Mice parenterally immunized with recombinant P vivax MSPI 19 in the presence of FliC, either admixed or genetically linked, elicited strong and long-lasting MSP1 (19)-specific systemic antibody responses with a prevailing IgG1 subclass response. Incorporation of another TLR agonist, CpG ODN 1826, resulted in a more balanced response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response measured by interferon-gamma secretion. Finally, we show that MSPI 19-specific antibodies recognized the native protein expressed on the surface of P. vivax parasites harvested from infected humans. The present report proposes a new class of malaria vaccine formulation based on the use of malaria antigens and the innate immunity agonist FliC. it contains intrinsic adjuvant properties and enhanced ability to induce specific humoral and cellular immune responses when administered alone or in combination with other adjuvants. (C) 2008 Elsevier Ltd. All rights reserved.

Immunogenic properties of a recombinant fusion protein containing the C-terminal 19 kDa of Plasmodium falciparum merozoite surface protein-1 and the innate immunity agonist FliC flagellin of Salmonella Typhimurium

In a recent study, we demonstrated the immunogenic properties of a new malaria vaccine polypeptide based on a 19 kDa C-terminal fragment of the merozoite surface protein-1 (MSP1(19)) from Plasmodium vivax and an innate immunity agonist, the Salmonella enterica serovar Typhimurium flagellin (FliC). Herein, we tested whether the same strategy, based on the MSP1(19) component of the deadly malaria parasite Plasmodium falciparum, could also generate a fusion polypeptide with enhanced immunogenicity. The His(6)FliC-MSP1(19) fusion protein was expressed from a recombinant Escherichia coil and showed preserved in vitro TLR5-binding activity. In contrast to animals injected with His(6)MSP1(19), mice subcutaneously immunised with the recombinant His6FliC-MSP1(19) developed strong MSP1(19)-specific systemic antibody responses with a prevailing IgG1 subclass. Incorporation of other adjuvants, such as CpG ODN 1826, complete and incomplete Freund`s adjuvants or Quil-A, improved the IgG responses after the second, but not the third, immunising dose. It also resulted in a more balanced IgG subclass response, as evaluated by the IgG1/IgG2c ratio, and higher cell-mediated immune response, as determined by the detection of antigen-specific interferon-gamma secretion by immune spleen cells. MSP(19)-specific antibodies recognised not only the recombinant protein, but also the native protein expressed on the surface of P. falciparum parasites. Finally, sera from rabbits immunised with the fusion protein alone inhibited the in vitro growth of three different P. falciparum strains. In summary, these results extend our previous observations and further demonstrate that fusion of the innate immunity agonist FliC to Plasmodium antigens is a promising alternative to improve their immunogenicity. (c) 2010 Elsevier Ltd. All rights reserved.

Evolutionary placement of Xanthomonadales based on conserved protein signature sequences

Xanthomonadales comprises one of the largest phytopathogenic bacterial groups, and is currently classified within the gamma-proteobacteria. However, the phylogenetic placement of this group is not clearly resolved, and the results of different studies contradict one another. In this work, the evolutionary position of Xanthomonadales was determined by analyzing the presence of shared insertions and deletions (INDELs) in highly conserved proteins. Several distinctive insertions found in most of the members of the gamma-proteobacteria are absent in Xanthomonadales and groups such as Legionelalles, Chromatiales, Methylococcales, Thiotrichales and Cardiobacteriales. These INDELs were most likely introduced after the branching of Xanthomonadales from most of the gamma-proteobacteria and provide evidence for the phylogenetic placement of the early gamma-proteobacteria. Moreover, other proteins contain insertions exclusive to the Xanthomonadales order, confirming that this is a monophyletic group and provide important specific genetic markers. Thus, the data presented clearly support the Xanthomonadales group as an independent subdivision, and constitute one of the deepest branching lineage within the gamma-proteobacteria clade. (C) 2009 Elsevier Inc. All rights reserved.

Endurance training activates AMP-activated protein kinase, increases expression of uncoupling protein 2 and reduces insulin secretion from rat pancreatic islets

Endurance exercise is known to enhance peripheral insulin sensitivity and reduce insulin secretion. However, it is unknown whether the latter effect is due to the reduction in plasma substrate availability or alterations in beta-cell secretory machinery. Here, we tested the hypothesis that endurance exercise reduces insulin secretion by altering the intracellular energy-sensitive AMP-activated kinase (AMPK) signaling pathway. Male Wistar rats were submitted to endurance protocol training one, three, or five times per week, over 8 weeks. After that, pancreatic islets were isolated, and glucose-induced insulin secretion (GIIS), glucose transporter 2 (GLUT2) protein content, total and phosphorylated calmodulin kinase kinase (CaMKII), and AMPK levels as well as peroxisome proliferator-activated receptor-gamma coactivator-1-alpha (PGC-1 alpha) and uncoupling protein 2 (UCP2) content were measured. After 8 weeks, chronic endurance exercise reduced GIIS in a dose-response manner proportionally to weekly exercise frequency. Contrariwise, increases in GLUT2 protein content, CaMKII and AMPK phosphorylation levels were observed. These alterations were accompanied by an increase in UCP2 content, probably mediated by an enhancement in PGC-1 alpha protein expression. In conclusion, chronic endurance exercise induces adaptations in beta-cells leading to a reduction in GIIS, probably by activating the AMPK signaling pathway. Journal of Endocrinology (2011) 208, 257-264

Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB