Targeting malaria with polyamines

During the asexual cycle of Plasmodium falciparum within the host erythrocyte, the parasite induces a stage-dependent elevation in the levels of polyamines by increased metabolism and uptake of extracellular pools. Polyamine amides of N-methylanthranilic acid have been synthesized which have embedded within them putrescine, spermidine, or spermine and from a charge perspective mimic natural polyamines. The interaction of these polyamine conjugates with human erythrocytes infected with malaria is described using fluorescent microscopy. The fluorescent spermine mimic was the only probe to show measurable intracellular accumulation. This was observed in late stage development but not in the ring stages or in uninfected erythrocytes.     

Structure of a synthetic glucose derived advanced glycation end product that is immunologically cross-reactive with its naturally occurring counterparts

Glucose reacts nonenzymatically with the amino groups of proteins to form stable, cross-linking adducts called advanced glycation end products or AGEs. While several lines of evidence have established that AGEs accumulate in tissues and contribute to the pathological sequelae of diabetes and aging, the identity of the major cross-link(s) that forms in vivo has remained enigmatic. This has been considered to be due to the labile nature and to the low fluorescence properties of this cross-link, despite the fact that fluorescence has been generally associated with AGE formation in vivo. Accordingly, the few AGE adducts that have been isolated thus far from proteins in vivo or from model systems in vitro have been found to account for only a fraction of the glucose-derived cross-links that occur in tissues. This situation has been further underscored by the development of a well-characterized class of antibodies that recognize in vivo AGEs but which fail to react with the structurally defined AGEs that have been identified to date. This particular class of anti-AGE antibodies has proven valuable in the quantification of AGE-modified forms of hemoglobin, low-density lipoprotein (LDL), and beta-amyloid peptide, and can provide prognostic information on the course of certain diabetic complications. To obtain insight into the structure of this immunoreactive, AGE adduct, we used an anti-AGE antibody (RU) as a probe to isolate novel AGE(s) that formed within a reaction mixture of glucose and the model glycation substrate, N(alpha)-CBZ-Arg-Lys. HPLC purification of an immunoreactive fraction that accumulated in this preparation showed the presence of a compound that was determined by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) to be a stable, imidazole-based cross-link (termed arginine-lysine imidazole or ALI). The properties of ALI, immunoreactivity, acid-lability, nonfluorescence, and inhibition of formation by aminoguanidine, suggest that ALI is likely to typify an important class of the AGE cross-links that form in vivo.     

Shoot meristem: an ideal explant for Zea mays L. transformation.

We report on a rapid high-frequency somatic embryogenesis and plant regeneration protocol for Zea mays. Maize plants were regenerated from complete shoot meristem (3-4 mm) explants via organogenesis and somatic embryogenesis. In organogenesis, the shoot meristems were directly cultured on a high-cytokinin medium comprising 5-10 mg x L(-1) 6-benzylaminopurine (BAP). The number of multiple shoots produced per meristem varied from six to eight Plantlet regeneration through organogenesis resulted in just four weeks. Callus was induced in five days of incubation on an auxin-modified Murashige and Skoog (MS) medium. Prolific callus, with numerous somatic embryos, developed within 3-4 weeks when cultured on an auxin medium containing 5 mg 2,4-dichlorophenoxyacetic acid x L(-1). The number of multiple shoots varied from three to six per callus. Using R23 (Pioneer, Hi-Bred, Johnston, Iowa), the frequency of callus induction was consistently in excess of 80% and plant regeneration ranged between 47 and 64%. All regenerated plantlets survived in the greenhouse and produced normal plants. Each transgenic plant produced leaves, glumes, and anthers that uniformly expressed green fluorescent protein (GFP). The GFP gene segregated in the pollen. Based on this data it is concluded that the transgenics arose from single-cell somatic embryos. The rate of transfer DNA (T-DNA) transfer to complete shoot meristems of Zea mays was high on the auxin medium and was independent of using super-virulent strains of Agrobacterium.     

Nicotinic acetylcholine receptor agonists attenuate septic acute kidney injury in mice by suppressing inflammation and proteasome activity

Sepsis is one of the leading causes of acute kidney injury (AKI). Septic patients who develop acute kidney injury (AKI) are at increased risk of death. To date there is no effective treatment for AKI or septic AKI. Based on their anti-inflammatory properties, we examined the effects of nicotinic acetylcholine receptor agonists on renal damage using a mouse model of lipopolysaccharide (LPS)-induced AKI where localized LPS promotes inflammation-mediated kidney damage. Administration of nicotine (1 mg/kg) or GTS-21 (4 mg/kg) significantly abrogated renal leukocyte infiltration (by 40%) and attenuated kidney injury. These renoprotective effects were accompanied by reduced systemic and localized kidney inflammation during LPS-induced AKI. Consistent with these observations, nicotinic agonist treatment significantly decreased renal IκBα degradation and NFκB activation during LPS-induced AKI. Treatment of human kidney cells with nicotinic agonists, an NFκB inhibitor (Bay11), or a proteasome inhibitor (MG132) effectively inhibited their inflammatory responses following stimulation with LPS or TNFα. Renal proteasome activity, a major regulator of NFκB-mediated inflammation, was enhanced by approximately 50% during LPS-induced AKI and elevated proteasome activity was significantly blunted by nicotinic agonist administration in vivo. Taken together, our results identify enhanced renal proteasome activity during LPS-induced AKI and the suppression of both proteasome activity and inflammation by nicotinic agonists to attenuate LPS-induced kidney injury.     

Semapimod Sensitizes Glioblastoma Tumors to Ionizing Radiation by Targeting Microglia

Glioblastoma is the most malignant and lethal form of astrocytoma, with patients having a median survival time of approximately 15 months with current therapeutic modalities. It is therefore important to identify novel therapeutics. There is mounting evidence that microglia (specialized brain-resident macrophages) play a significant role in the development and progression of glioblastoma tumors. In this paper we show that microglia, in addition to stimulating glioblastoma cell invasion, also promote glioblastoma cell proliferation and resistance to ionizing radiation in vitro. We found that semapimod, a drug that selectively interferes with the function of macrophages and microglia, potently inhibits microglia-stimulated GL261 invasion, without affecting serum-stimulated glioblastoma cell invasion. Semapimod also inhibits microglia-stimulated resistance of glioblastoma cells to radiation, but has no significant effect on microglia-stimulated glioblastoma cell proliferation. We also found that intracranially administered semapimod strongly increases the survival of GL261 tumor-bearing animals in combination with radiation, but has no significant benefit in the absence of radiation. In conclusion, our observations indicate that semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia and/or infiltrating macrophages.     

The HIV Protease Inhibitor Saquinavir Inhibits HMGB1-Driven Inflammation by Targeting the Interaction of Cathepsin V with TLR4/MyD88

Extracellular high-mobility group box 1 (HMGB1) (disulfide form), via activation of toll-like receptor 4 (TLR4)-dependent signaling, is a strong driver of pathologic inflammation in both acute and chronic conditions. Identification of selective inhibitors of HMGB1-TLR4 signaling could offer novel therapies that selectively target proximal endogenous activators of inflammation. A cell-based screening strategy led us to identify first generation HIV-protease inhibitors (PI) as potential inhibitors of HMGB1-TLR4 driven cytokine production. Here we report that the first-generation HIV-PI saquinavir (SQV), as well as a newly identified mammalian protease inhibitor STO33438 (334), potently block disulfide HMGB1-induced TLR4 activation, as assayed by the production of TNF-α by human monocyte-derived macrophages (THP-1). We further report on the identification of mammalian cathepsin V, a protease, as a novel target of these inhibitors. Cellular as well as recombinant protein studies show that the mechanism of action involves a direct interaction between cathepsin V with TLR4 and its adaptor protein MyD88. Treatment with SQV, 334 or the known cathepsin inhibitor SID26681509 (SID) significantly improved survival in murine models of sepsis and reduced liver damage following warm liver ischemia/reperfusion (I/R) models, both characterized by strong HMGB1-TLR4 driven pathology. The current study demonstrates a novel role for cathepsin V in TLR4 signaling and implicates cathepsin V as a novel target for first-generation HIV-PI compounds. The identification of cathepsin V as a target to block HMGB1-TLR4-driven inflammation could allow for a rapid transition of the discovery from the bench to the bedside. Disulfide HMGB1 drives pathologic inflammation in many models by activating signaling through TLR4. Cell-based screening identified the mammalian protease cathepsin V as a novel therapeutic target to inhibit TLR4-mediated inflammation induced by extracellular HMGB1 (disulfide form). We identified two protease inhibitors (PIs) that block cathepsin V and thereby inhibit disulfide HMGB1-induced TLR4 activation: saquinavir (SQV), a first-generation PI targeting viral HIV protease and STO33438 (334), targeting mammalian proteases. We discovered that cathepsin V binds TLR4 under basal and HMGB1-stimulated conditions, but dissociates in the presence of SQV over time. Thus cathepsin V is a novel target for first-generation HIV PIs and represents a potential therapeutic target of pathologic inflammation.     

Macrophage migration inhibitory factor (MIF) is necessary for progression of autoimmune diabetes mellitus

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine of the innate immune system that plays a major role in the induction of immunoinflammatory responses. To examine the role of endogenous MIF in the pathogenesis of type 1 diabetes (TID) we evaluated the effects of administration of neutralizing anti-MIF antibodies to NOD mice with accelerated forms of diabetes induced by injection of cyclophosphamide or by transfer of diabetogenic spleen cells. Both accelerated forms of diabetes were markedly reduced by anti-MIF antibody. Furthermore, MIF-deficient (MIF(-/-)) mice were less susceptible to the induction of immunoinflammatory diabetes, insulitis and apoptosis within the endocrine pancreas by multiple low doses of streptozotocin (MLD-STZ) than genetically matched wild type (WT) mice. MIF deficiency resulted in lower proliferation and lymphocyte adhesion, as well as reduced production from the spleens and peritoneal cells of a variety of inflammatory mediators typically associated with development of the disease including IL-12, IL-23, TNF-alpha, and IL-1beta. Furthermore, MIF deletion affected the production of IL-18, TNF-alpha, IL-1beta, and iNOS in the islets of Langerhans. These data, along with the higher expression of IL-4 and TGF-beta observed in the periphery and in the pancreas of MLD-STZ-challenged MIF(-/-) mice as compared to WT controls suggest that MIF deficiency has induced an immune deviation towards protective type 2/3 response. These results suggest that MIF participates in T1D by controlling the functional activity of monocytes/macrophages and T cells and modulating their secretory capacity of pro- and anti-inflammatory molecules.     

Inhibition of HIV-1 nuclear import via schiff base formation with arylene bis(methylketone) compounds

Arylene bis(methylketone) compounds specifically block nuclear translocation of the HIV-1 pre-integration complex by forming Schiff-base adducts with contiguous lysines within nuclear localization signal.