Design, synthesis and biological evaluation of alkynyl-containing maleimide derivatives for the treatment of drug-resistant tuberculosis.

A series of alkynyl-containing maleimides with potent anti-tuberculosis (TB) activity was developed through a rigid group substitution strategy based on our previous study. Systematic optimization of the two side chains flanking the maleimide core led to new compounds with potent activity against Mycobacterium tuberculosis (MIC < 1 µg/mL) and low cytotoxicity (IC50 > 64 µg/mL). Among them, compound 29 not only possessed good activity against extensively drug-resistant TB and favorable hepatocyte stability, but also displayed good intracellular antimycobacterial activity in macrophages. This study lays a good foundation for identifying new alkynyl-containing maleimides as promising leads for treating drug-resistant TB.

Opuntin B, the antiviral protein isolated from prickly pear (Opuntia ficus-indica (L.) Miller) cladode exhibits ribonuclease activity.

An antiviral protein, designated Opuntin B, was purified from Prickly Pear (Opuntia ficus-indica (L.) Miller) Cladode by heat treatment of the extract, protein precipitation by ammonium sulfate treatment followed by ion-exchange chromatography. Assessment of enzymatic activity of the purified protein showed that it degrades total plant genomic RNA, while causing electrophoretic mobility shifting of Cucumber mosaic virus (CMV) RNAs. However, heat-denatured viral RNA became sensitive to degradation upon treatment with antiviral protein. Opuntin B had no DNase activity on native and heat-denatured apricot genomic DNA, and on PCR-amplified coat protein gene of CMV. Using CMV as prey protein and Opuntin B as bait protein, no interaction was found between the antiviral protein and viral coat protein in far western dot blot analysis.

Asiatic acid, an active substance of Centella asiatica, presynaptically depresses glutamate release in the rat hippocampus.

Inhibiting glutamate release can reduce neuronal excitability and is recognized as a key mechanism of anti-epileptic drugs. In this study, by using isolated nerve terminal (synaptosome) and slice preparations, we investigated the effect of asiatic acid, a triterpene isolated from Centella asiatica with antiepileptic activity, on glutamate release in the hippocampus of rats. In hippocampal synaptosomes, application of asiatic acid resulted in a concentration-dependent inhibition of 4-aminopyridine-evoked glutamate release. This inhibitory action was dependent on extracellular calcium, blocked by inhibiting the vesicular transporter, but was unaffected by inhibiting the glutamate transporter. In addition, asiatic acid decreased the 4-aminopyridine-induced increase in the intraterminal calcium and failed to alter the synaptosomal potential. Furthermore, the asiatic acid-mediated release inhibition was significantly suppressed by the N- and P/Q-type calcium channel inhibitor ω-conotoxin MVIIC or protein kinase C inhibitor GF109203X. Western blotting data in synaptosomes also revealed that asiatic acid reduced 4-aminopyridine-induced phosphorylation of protein kinase C. In hippocampal slices, asiatic acid decreased the frequencies of spontaneous excitatory postsynaptic currents without changing their amplitudes and glutamate-activated currents in CA3 pyramidal neurons. We also observed that asiatic acid significantly suppressed 4-aminopyridine-induced burst firing. These data suggest that, in rat hippocampal nerve terminals, asiatic acid attenuates the calcium influx via N- and P/Q-type calcium channels, subsequently suppressing protein kinase C activity and decreasing glutamate release.

Increased Inhibition Effect of Antrodin C from the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes), on A549 through Crosstalk between Apoptosis and Autophagy.

Antrodin C was obtained from Taiwanofungus camphoratus mycelia. The inhibition effect of antrodin C on A549 lung adenocarcinoma cells was evaluated by plate clone formation, wound healing, cell cycle, activated caspase-3, Bax, P53, Bcl-2, and RAPR activities as well as reactive oxygen species release. Plate clone formation assay revealed that antrodin C could significantly inhibit the viability of A549 cells in vitro. Wound healing assay revealed that cell migration was inhibited by exposure to antrodin C at concentrations of 50 and 80 µg/mL. Flow cytometry revealed that antrodin C increased the percentages of cells in the G0/G1 phase at concentrations of 50 and 80 µg/mL and the apoptosis was related to upregulation of caspase-3, Bax, P53 expression, downregulation of Bcl-2, RAPR expression, and the release of reactive oxygen species in the A549 cells. CQ or RAPA could significantly promote or inhibit the inhibition effect on A549 proliferation induced by antrodin C, which suggests that the autophagy played a cytoprotective role on inhibition proliferation of A549 induced by antrodin C. These results indicated that the combination of pro-apoptosis agents and anti-autophagy agents may be a useful strategy in enhancing the anticancer efficacy in non-small cell lung cancer.

New eremophilane-type sesquiterpenes and maleimide-bearing compounds from Carpesium abrotanoides L.

Two new eremophilane-type sesquiterpenes, carperemophilanes A and B (1-2), three new maleimide-bearing compounds, carpesiumaleimides A-C (3-5), along with a known sesquiterpene, carabrol (6), were isolated from the ethanol extract of Carpesium abrotanoides L. Their structures were elucidated by analysis of their NMR and MS data as well as by comparison with the literature. The absolute configuration of carperemophilane A (1) was determined by single-crystal X-ray diffraction analysis. All isolated compounds (1-6) were evaluated in vitro for cytotoxicity against two human cancer cell lines MDA-MB-231 and HGC-27 using the MTT method. Compounds 1, 2 and 6 showed cytotoxic activities with IC50 values ranging from 7.45 to 37.35 µM.

Berberine coated mannosylated liposomes curtail RANKL stimulated osteoclastogenesis through the modulation of GSK3ß pathway via upregulating miR-23a.

Drug-induced microRNAs manifest significant therapeutic approaches; however, such progress in the treatment of osteopathic disorders including osteoporosis and rheumatoid arthritis still remains obscure. Contrarily, non-specific drug delivery, at high doses, increases the risk of side effects and reduces drug therapeutic efficacy. Accordingly, the present study was designed to examine the therapeutic effect of berberine coated mannosylated liposomes (ML-BBR) on RANKL (100 ng/ml) stimulated bone marrow-derived monocytes/macrophages (BMMs) via altering miR-23a expression. Initial studies using confocal microscopy showed successful internalization of ML-BBR in RANKL stimulated BMMs. Treatment with ML-BBR abrogated the increased osteoclast formation in BMM cells via inhibiting phosphorylated glutathione synthase kinase beta (p-GSK3ß) mediated NFATc1 activation. Consequently, ML-BBR also attenuated the expression of bone-degrading enzymes (TRAP, cathepsin K and MMP-9) thereby inhibiting the bone resorptive activity of osteoclasts. Moreover, ML-BBR induced the expression levels of miR-23a at the gene level, which in turn attenuated GSK3ß/p-GSK3ß expression as confirmed via blotting analysis. Further miR-23a inhibition of the GSK3ß phosphorylation was confirmed using luciferase reporter assay. Comparatively, LY2090314 (GSK3ß inhibitor) treatment inhibited the protein level expression of GSK3ß/p-GSK3ß. However, LY2090314 treatment induced a basal level expression of miR-23a owing to the suggestion that ML-BBR has an influential role in upregulating miR-23a level to inhibit GSK-3ß phosphorylation. Cumulatively, our findings endorsed that preferential internalization of ML-BBR by BMMs effectively modulated the RANKL/p-GSK3ß pathway and curtailed the osteoclast-mediated bone erosion possibly through post-transcriptional gene silencing via miR-23a.

Natural Product-Based Pesticide Discovery: Design, Synthesis and Bioactivity Studies of N-Amino-Maleimide Derivatives.

Natural products are an important source of pesticide discovery. A series of N-amino-maleimide derivatives containing hydrazone group were designed and synthesized based on the structure of linderone and methyllinderone which were isolated from Lindera erythrocarpa Makino. According to the bioassay results, compounds 2 and 3 showed 60% inhibition against mosquito (Culex pipiens pallens) at 0.25 µg·mL−1. Furthermore, the results of antifungal tests indicated that most compounds exhibited much better antifungal activities against fourteen phytopathogenic fungi than linderone and methyllinderone and some compounds exhibited better antifungal activities than commercial fungicides (carbendazim and chlorothalonil) at 50 µg·mL−1. In particular, compound 12 exhibited broad-spectrum fungicidal activity (>50% inhibitory activities against 11 phytopathogenic fungi) and compounds 12 and 14 displayed 60.6% and 47.9% inhibitory activity against Rhizoctonia cerealis at 12.5 µg·mL−1 respectively. Furthermore, compound 17 was synthesized, which lacks N-substituent at maleimide and its poor antifungal activity against Sclerotinia sclerotiorum and Rhizoctonia cerealis at 50 µg·mL−1 showed that the backbone structure of N-amino-maleimide derivatives containing hydrazone group was important to the antifungal activity.

Onion (Allium cepa L.) peel extract (OPE) regulates human sperm motility via protein kinase C-mediated activation of the human voltage-gated proton channel.

Onion (Allium cepa L.) and quercetin protect against oxidative damage and have positive effects on multiple functional parameters of spermatozoa, including viability and motility. However, the associated underlying mechanisms of action have not yet been identified. The aim of this study was to investigate the effect of onion peel extract (OPE) on voltage-gated proton (Hv1) channels, which play a critical role in rapid proton extrusion. This process underlies a wide range of physiological processes, particularly male fertility. The whole-cell patch-clamp technique was used to record the changes in Hv1 currents in HEK293 cells transiently transfected with human Hv1 (HVCN1). The effects of OPE on human sperm motility were also analyzed. OPE significantly activated the outward-rectifying proton currents in a concentration-dependent manner, with an EC50 value of 30 µg/mL. This effect was largely reversible upon washout. Moreover, OPE induced an increase in the proton current amplitude and decreased the time constant of activation at 0 mV from 4.9 ± 1.7 to 0.6 ± 0.1 sec (n = 6). In the presence of OPE, the half-activation voltage (V1/2 ) shifted in the negative direction, from 20.1 ± 5.8 to 5.2 ± 8.7 mV (n = 6), but the slope was not significantly altered. The OPE-induced current was profoundly inhibited by 10 µm Zn2+ , the most potent Hv1 channel inhibitor, and was also inhibited by treatment with GF109203X, a specific protein kinase C (PKC) inhibitor. Furthermore, sperm motility was significantly increased in the OPE-treated groups. OPE exhibits protective effects on sperm motility, at least partially via regulation of the proton channel. Moreover, similar effects were exerted by quercetin, the major flavonoid in OPE. These results suggest OPE, which is rich in the potent Hv1 channel activator quercetin, as a possible new candidate treatment for human infertility.

Activation of GSK-3 disrupts cholinergic homoeostasis in nucleus basalis of Meynert and frontal cortex of rats.

The cholinergic impairment is an early marker in Alzheimer's disease (AD), while the mechanisms are not fully understood. We investigated here the effects of glycogen synthase kinse-3 (GSK-3) activation on the cholinergic homoeostasis in nucleus basalis of Meynert (NBM) and frontal cortex, the cholinergic enriched regions. We activated GSK-3 by lateral ventricular infusion of wortmannin (WT) and GF-109203X (GFX), the inhibitors of phosphoinositol-3 kinase (PI3-K) and protein kinase C (PKC), respectively, and significantly decreased the acetylcholine (ACh) level via inhibiting choline acetyl transferase (ChAT) rather than regulating acetylcholinesterase (AChE). Neuronal axonal transport was disrupted and ChAT accumulation occurred in NBM and frontal cortex accompanied with hyperphosphorylation of tau and neurofilaments. Moreover, ChAT expression decreased in NBM attributing to cleavage of nuclear factor-κB/p100 into p52 for translocation into nucleus to lower ChAT mRNA level. The cholinergic dysfunction could be mimicked by overexpression of GSK-3 and rescued by simultaneous administration of LiCl or SB216763, inhibitors of GSK-3. Our data reveal the molecular mechanism that may underlie the cholinergic impairments in AD patients.

Bisindolylmaleimide alkaloid BMA-155Cl induces autophagy and apoptosis in human hepatocarcinoma HepG-2 cells through the NF-κB p65 pathway.

Bisindolylmaleimides, a series of derivatives of a PKC inhibitor staurosporine, exhibit potential as anti-cancer drugs and have received considerable attention in clinical trials. This study aims to investigate the effects of a bisindolylmaleimide alkaloid 155Cl (BMA-155Cl) with a novel structure on autophagy and apoptosis in human hepatocarcinoma HepG-2 cells in vitro and in vivo. The cell poliferation was assessed with a MTT assay. Autophagy was evaluated by MDC staining and TEM analysis. Apoptosis was investigated using Annexin V-FITC/PI and DAPI staining. The antitumor effects were further evaluated in nude mice bearing HepG-2 xenografts, which received BMA-155Cl (10, 20 mg/kg, ip) for 18 days. Autophagy- and apoptosis-associated proteins and their mRNA levels were examined with Western blotting, immunohistochemistry, and RT-PCR. BMA-155Cl (2.5-20 µmol/L) inhibited the growth of HepG-2 cells with IC50 values of 16.62±1.34, 12.21±0.83, and 8.44±1.82 µmol/L at 24, 48, and 72 h, respectively. Furthermore, BMA-155Cl (5-20 µmol/L) dose-dependently induced autophagy and apoptosis in HepG-2 cells. The formation of autophagic vacuoles was induced by BMA-155Cl (10 µmol/L) at approximately 6 h and peaked at approximately 15 h. Pretreatment with 3-MA potentiated BMA-155Cl-mediated apoptotic cell death. This compound dose-dependently increased the mRNA and protein levels of Beclin-1, NF-κB p65, p53, and Bax, but decreased the expression of IκB and Bcl-2. Pretreatment with BAY 11-7082, a specific inhibitor of NF-κB p65, blocked BMA-155Cl-induced expression of autophagy- and apoptosis-associated proteins. BMA-155Cl administration effectively suppressed the growth of HepG-2 xenografts in vivo, and increased the protein expression levels of LC3B, Beclin-1, NF-κB p65, and Bax in vivo. We conclude that the NF-κB p65 pathway is involved in BMA-155Cl-triggered autophagy, followed by apoptosis in HepG-2 cells in vitro and in vivo. Hence, BMA-155Cl could be a promising pro-apoptotic candidate for developing as a novel anti-cancer drug.

[MORPHOFUNCTIONAL STATE OF BLOOD CELLS AFTER CHRONIC EXPOSURE OF THE PROTEIN KINASES INHIBITOR MALEIMIDE DERIVATIVE].

The effect of the protein kinases inhibitor maleimide derivative (MI-1, 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione), inhibitor of VEGF-R1,2,3, FGF-R1, EGF-R(h), PDK1, Src(h), Syk(h), YES, ZAP70 et al. with antineoplastic activity, on blood cells parameters of rats after chronic exposure has been studied. Administration of MI-1 at doses 0.027 and 2.7 mg/kg (suppress colon carcinogenesis) for 20 and 26 weeks does not affect the morphofunctional state of red blood cells in healthy rats. This is confirmed by the lack of differences in the concentration of hemoglobin in blood, red blood cells count, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, hematocrit and mean corpuscular volume, and the number of reticulocytes in blood after 20 and 26 weeks of exposure compared with the control group. MI-1 at indicated doses does not influence total leukocytes count and content (eosinophilic and neutrophilic granulocytes, lymphocytes, monocytes) and does not inhibit thrombocytopoiesis (platelet count remains unchanged). No negative effect of MI-1 on hematopoiesis is not limited (by the hemopoietic system) use of this compound as a potential antitumor drug

Prokineticin 2 facilitates mechanical allodynia induced by α,ß-methylene ATP in rats.

Prokineticin 2 (PK2), a new chemokine, causes mechanical hypersensitivity in the rat hind paw, but little is known about the molecular mechanism. Here, we have found that ionotropic P2X receptor is essential to mechanical allodynia induced by PK2. First, intraplantar injection of high dose (3 or 10 pmol) of PK2 significantly increased paw withdrawal response frequency (%) to innocuous mechanical stimuli (mechanical allodynia). And the mechanical allodynia induced by PK2 was prevented by co-administration of TNP-ATP, a selective P2X receptor antagonist. Second, although low dose (0.3 or 1 pmol) of PK2 itself did not produce an allodynic response, it significantly facilitated the mechanical allodynia evoked by intraplantar injection of α,ß-methylene ATP (α,ß-meATP). Third, PK2 concentration-dependently potentiated α,ß-meATP-activated currents in rat dorsal root ganglion (DRG) neurons. Finally, PK2 receptors and intracellular signal transduction were involved in PK2 potentiation of α,ß-meATP-induced mechanical allodynia and α,ß-meATP-activated currents, since the potentiation were blocked by PK2 receptor antagonist PKRA and selective PKC inhibitor GF 109203X. These results suggested that PK2 facilitated mechanical allodynia induced by α,ß-meATP through a mechanism involved in sensitization of cutaneous P2X receptors expressed by nociceptive nerve endings.

Physiological, pharmacological and behavioral evidence for a TRPA1 channel that can elicit defensive responses in the medicinal leech.

Transient receptor potential ankyrin subtype 1 (TRPA1) channels are chemosensitive to compounds such as allyl isothiocyanate (AITC, the active component of mustard oil) and other reactive electrophiles and may also be thermodetectors in many animal phyla. In this study, we provide the first pharmacological evidence of a putative TRPA1-like channel in the medicinal leech. The leech's polymodal nociceptive neuron was activated by both peripheral and central application of the TRPA1 agonist AITC in a concentration-dependent manner. Responses to AITC were inhibited by the selective TRPA1 antagonist HC030031, but also by the TRPV1 antagonist SB366791. Other TRPA1 activators - N-methylmaleimide (NMM) and cinnamaldehyde (CIN) - also activated this nociceptive neuron, although HC030031 only inhibited the effects of NMM. The polymodal nociceptive neurons responded to moderately cold thermal stimuli (<17°C) and these responses were blocked by HC030031. AITC sensitivity was also found in the pressure-sensitive sensory neurons and was blocked by HC030031, but not by SB366791. AITC elicited a nocifensive withdrawal of the posterior sucker in a concentration-dependent manner that could be attenuated with HC030031. Peripheral application of AITC in vivo also produced swimming-like behavior that was attenuated by HC030031. These results suggest the presence of a TRPA1-like channel in the medicinal leech nervous system that responds to cold temperatures and may interact with the leech TRPV-like channel.

Procyanidin trimer C1 derived from Theobroma cacao reactivates latent human immunodeficiency virus type 1 provirus.

Despite remarkable advances in combination antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) infection remains incurable due to the incomplete elimination of the replication-competent virus, which persists in latent reservoirs. Strategies for targeting HIV reservoirs for eradication that involves reactivation of latent proviruses while protecting uninfected cells by cART are urgently needed for cure of HIV infection. We screened medicinal plant extracts for compounds that could reactivate the latent HIV-1 provirus and identified a procyanidin trimer C1 derived from Theobroma cacao as a potent activator of the provirus in human T cells latently infected with HIV-1. This reactivation largely depends on the NF-κB and MAPK signaling pathways because either overexpression of a super-repressor form of IκBα or pretreatment with a MEK inhibitor U0126 diminished provirus reactivation by C1. A pan-PKC inhibitor significantly blocked the phorbol ester-induced but not the C1-induced HIV-1 reactivation. Although C1-induced viral gene expression persisted for as long as 48 h post-stimulation, NF-κB-dependent transcription peaked at 12 h post-stimulation and then quickly declined, suggesting Tat-mediated self-sustainment of HIV-1 expression. These results suggest that procyanidin C1 trimer is a potential compound for reactivation of latent HIV-1 reservoirs.

Acute administration of n-3 rich triglyceride emulsions provides cardioprotection in murine models after ischemia-reperfusion.

Dietary n-3 fatty acids (FAs) may reduce cardiovascular disease risk. We questioned whether acute administration of n-3 rich triglyceride (TG) emulsions could preserve cardiac function and decrease injury after ischemia/reperfusion (I/R) insult. We used two different experimental models: in vivo, C57BL/6 mice were exposed to acute occlusion of the left anterior descending coronary artery (LAD), and ex-vivo, C57BL/6 murine hearts were perfused using Langendorff technique (LT). In the LAD model, mice treated with n-3 TG emulsion (1.5 g/kg body weight), immediately after ischemia and 1 h later during reperfusion, significantly reduced infarct size and maintained cardiac function (p<0.05). In the LT model, administration of n-3 TG emulsion (300 mg TG/100 ml) during reperfusion significantly improved functional recovery (p<0.05). In both models, lactate dehydrogenase (LDH) levels, as a marker of injury, were significantly reduced by n-3 TG emulsion. To investigate the mechanisms by which n-3 FAs protects hearts from I/R injury, we investigated changes in key pathways linked to cardioprotection. In the ex-vivo model, we showed that n-3 FAs increased phosphorylation of AKT and GSK3ß proteins (p<0.05). Acute n-3 TG emulsion treatment also increased Bcl-2 protein level and reduced an autophagy marker, Beclin-1 (p<0.05). Additionally, cardioprotection by n-3 TG emulsion was linked to changes in PPARγ protein expression (p<0.05). Rosiglitazone and p-AKT inhibitor counteracted the positive effect of n-3 TG; GSK3ß inhibitor plus n-3 TG significantly inhibited LDH release. We conclude that acute n-3 TG injection during reperfusion provides cardioprotection. This may prove to be a novel acute adjunctive reperfusion therapy after treating patients with myocardial infarction.

Lithium attenuates lipopolysaccharide-induced hypothermia in rats.

BACKGROUND: Changes in body temperature are common features among patients with sepsis and septic shock. Similarly, systemic administration of bacterial endotoxin (lipopolysaccharide, LPS) to rats leads to an initial hypothermia followed by elevation in body temperature. These changes in body temperature are accompanied by increased levels of prostaglandin E2 (PGE2) in the hypothalamus. OBJECTIVE: This study examined the effects of lithium and SB216763 - two different glycogen synthase kinase (GSK)-3 inhibitors - on LPS-induced changes in body temperature and hypothalamic PGE2 levels in endotoxemic rats. MATERIALS AND METHODS: Endotoxemia was induced by intraperitoneal injection of LPS (10 mg/kg). Lithium (100 mg/kg) and SB216763 (5 mg/kg) were administered at 2 h before LPS. Body temperature and mortality were monitored during 48 h after LPS injection. In another protocol, rats were sacrificed at 2 h post LPS injection and then, blood, liver and hypothalamus were extracted for inflammatory mediators determination. RESULTS: Lithium but not SB216763 significantly reduced LPS-induced hypothermia, while both compounds did not alter the subsequent elevation in body temperature. Moreover, only lithium significantly reduced hypothalamic PGE2 levels. On the other hand, both compounds significantly reduced plasma, hepatic and hypothalamic tumor necrosis factor-α and decreased plasma PGE2 levels. Both compounds did not alter LPS-induced mortality. CONCLUSIONS: These results suggest that the attenuation of LPS-induced hypothermia by lithium may derive from its reduction of hypothalamic PGE2 levels.

Cell-based assays for Parkinson's disease using differentiated human LUHMES cells.

AIM: Lund human mesencephalic (LUHMES) cells can be differentiated to post-mitotic cells with biochemical, morphological and functional features of dopaminergic (DAergic) neurons. Given the limited scale of primary DAergic neuron culture, we developed differentiated LUHMES cell-based cytotoxicity assays for identifying neuroprotective agents for Parkinson's disease (PD). METHODS: LUHMES cells were incubated in a differentiation medium containing cAMP and GDNF for 6 d, and then differentiated cells were treated with MPP(+) or infected with baculovirus containing α-synuclein. Cytotoxicity was determined by measuring intracellular ATP levels and caspase 3/7 activity in the cells. DAergic neuron-specific marker protein and mRNA levels in the cells were analyzed using Western blotting and RT-PCR, respectively. RESULTS: LUHMES cells grew extensive neurites and became post-mitotic neuron-like cells during differentiation period, and three DAergic neuron markers TH, DAT and Nurr1 exhibited different expression profiles. MPP(+) dose-dependently reduced ATP levels in the cells with an IC50 value of 65 µmol/L. MPP(+) (80 µmol/L) significantly increased caspase 3/7 activity in the cells. Both the CDK inhibitor GW8510 and the GSK3ß inhibitor SB216763 effectively rescued MPP(+)-induced reduction of ATP levels with EC50 values of 12 and 205 nmol/L, respectively. Overexpression of α-synuclein also significantly decreased intracellular ATP levels and increased caspase 3/7 activity in the cells. GW8510 and SB216763 effectively rescued α-synuclein overexpression-induced reduction of ATP levels, whereas GW8510, but not SB216763, ameliorated α-synuclein overexpression-induced increase of caspase 3/7 activity. CONCLUSION: MPP(+)- and α-synuclein overexpression-induced cytotoxicity of differentiated LUHMES cells may serve as good alternative systems for identifying neuroprotective compounds for PD.

Inhibiting glycogen synthase kinase-3 mitigates the hematopoietic acute radiation syndrome in mice.

Exposure to a nuclear accident or radiological attack can cause death from acute radiation syndrome (ARS), which results from radiation injury to vital organs such as the hematopoietic system. However, the U.S. Food and Drug Administration (FDA) has not approved any medical countermeasures for this specific purpose. With growing concern over nuclear terrorism, there is an urgent need to develop small molecule deliverables that mitigate mortality from ARS. One emerging modulator of hematopoietic stem/progenitor cell (HSPC) activity is glycogen synthase kinase-3 (GSK-3). The inhibition of GSK-3 has been shown to augment hematopoietic repopulation in mouse models of bone marrow transplantation. In this study, we performed an in vitro screen using irradiated bone marrow mononuclear cells (BM-MNCs) to test the effects of four GSK-3 inhibitors: CHIR99021; 6-Bromoindirubin-3'-oxime (BIO); SB415286; and SB216763. This screen showed that SB216763 significantly increased the frequency of c-Kit(+) Lin(-) Sca1(+) (KLS) cells and hematopoietic colony-forming cells in irradiated BM-MNCs. Importantly, administration of a single dose of SB216763 to C57BL/6J mice by subcutaneous injection 24 h after total-body irradiation significantly improved hematopoietic recovery and mitigated hematopoietic ARS. Collectively, our results demonstrate that the GSK-3 inhibitor SB216763 is an effective medical countermeasure against acute radiation injury of the hematopoietic system.

Phosphorylation of amyloid precursor protein at threonine 668 is essential for its copper-responsive trafficking in SH-SY5Y neuroblastoma cells.

Amyloid precursor protein (APP) undergoes post-translational modification, including O- and N-glycosylation, ubiquitination, and phosphorylation as it traffics through the secretory pathway. We have previously reported that copper promotes a change in the cellular localization of APP. We now report that copper increases the phosphorylation of endogenous APP at threonine 668 (Thr-668) in SH-SY5Y neuronal cells. The level of APPT668-p (detected using a phospho-site-specific antibody) exhibited a copper-dependent increase. Using confocal microscopy imaging we demonstrate that the phospho-deficient mutant, Thr-668 to alanine (T668A), does not exhibit detectable copper-responsive APP trafficking. In contrast, mutating a serine to an alanine at residue 655 does not affect copper-responsive trafficking. We further investigated the importance of the Thr-668 residue in copper-responsive trafficking by treating SH-SY5Y cells with inhibitors for glycogen synthase kinase 3-ß (GSK3ß) and cyclin-dependent kinases (Cdk), the main kinases that phosphorylate APP at Thr-668 in neurons. Our results show that the GSK3ß kinase inhibitors LiCl, SB 216763, and SB 415286 prevent copper-responsive APP trafficking. In contrast, the Cdk inhibitors Purvalanol A and B had no significant effect on copper-responsive trafficking in SH-SY5Y cells. In cultured primary hippocampal neurons, copper promoted APP re-localization to the axon, and this effect was inhibited by the addition of LiCl, indicating that a lithium-sensitive kinase(s) is involved in copper-responsive trafficking in hippocampal neurons. This is consistent with APP axonal transport to the synapse, where APP is involved in a number of functions. We conclude that copper promotes APP trafficking by promoting a GSK3ß-dependent phosphorylation in SH-SY5Y cells.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats.

BACKGROUND: Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage. STUDY DESIGN AND METHODS: The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage. We exposed rats to exchange transfusion (ET; 50% blood volume and isovolemic replacement with Hextend [negative colloid control], MP4OX [PEGylated HBOC with high oxygen affinity], and ααHb [αα-cross-linked HBOC with low oxygen affinity; n=4-6/group]). Sham rats acted as control. Animals were euthanized at 2, 6, and 12 hours after ET; brain tissue was harvested and processed for analysis. RESULTS: In MP4OX animals, the number of neurons that overexpressed the hypoxia-inducible factor (HIF)-1α was higher than in ααHb, particularly at the early time points. In addition, MP4OX was associated with greater phosphorylation of protein kinase B (Akt), a well-known cytoprotective factor. Indeed, the degree of apoptosis, measured as terminal deoxynucleotidyl transferase-positive neurons and caspase-3 cleavage, ranked in order of MP4OX < Hextend < ααHb. CONCLUSION: Even though both HBOCs showed increased levels of HIF-1α compared to shams or Hextend-treated animals, differences in signaling events resulted in very different outcomes for the two HBOCs. ααHb-treated brain tissue showed significant neuronal damage, measured as apoptosis. This was in stark contrast to the protection seen with MP4OX, apparently due to recruitment of Akt and neuronal specific HIF-1α pathways.