Chronic N-methyl-D-aspartate administration increases the turnover of arachidonic acid within brain phospholipids of the unanesthetized rat

Whereas antibipolar drug administration to rats reduces brain arachidonic acid turnover, excessive N-methyl-d-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms and may increase arachidonic acid turnover in rat brain phospholipids. To determine whether chronic NMDA would increase brain arachidonic acid turnover, rats were daily administered NMDA (25 mg/kg, ip) or vehicle for 21 days. In unanesthetized rats, on day 21, [1-(14)C]arachidonic acid was infused intravenously and arterial blood plasma was sampled until the animal was euthanized at 5 min and its microwaved brain was subjected to chemical and radiotracer analysis. Using equations from our in vivo fatty acid model, we found that compared with controls, chronic NMDA increased the net rate of incorporation of plasma unesterified arachidonic acid into brain phospholipids (25-34%) as well as the turnover of arachidonic acid within brain phospholipids (35-58%). These changes were absent at 3 h after a single NMDA injection. The changes, opposite to those after chronic administration of antimanic drugs to rats, suggest that excessive NMDA signaling via arachidonic acid may be a model of upregulated arachidonic acid turnover in brain phospholipids.     

Chronic NMDA Administration Increases Neuroinflammatory Markers in Rat Frontal Cortex: Cross-Talk Between Excitotoxicity and Neuroinflammation

Chronic N-Methyl-d-aspartate (NMDA) administration, a model of excitotoxicity, and chronic intracerebroventricular lipopolysaccharide infusion, a model of neuroinflammation, are reported to upregulate arachidonic acid incorporation and turnover in rat brain phospholipids as well as enzymes involved in arachidonic acid metabolism. This suggests cross-talk between signaling pathways of excitotoxicity and of neuroinflammation, involving arachidonic acid. To test whether chronic NMDA administrations to rats can upregulate brain markers of neuroinflammation, NMDA (25 mg/kg i.p.) or vehicle (1 ml saline/kg i.p.) was administered daily to adult male rats for 21 days. Protein and mRNA levels of cytokines and other inflammatory markers were measured in the frontal cortex using immunoblot and real-time PCR. Compared with chronic vehicle, chronic NMDA significantly increased protein and mRNA levels of interleukin-1beta, tumor necrosis factor alpha, glial fibrillary acidic protein and inducible nitric oxide synthase. Chronic NMDA receptor overactivation results in increased levels of neuroinflammatory markers in the rat frontal cortex, consistent with cross-talk between excitotoxicity and neuroinflammation. As both processes have been reported in a number of human brain diseases, NMDA receptor inhibitors might be of use in treating neuroinflammation in these diseases.     

Translation inhibition during cell cycle arrest and apoptosis: Mcl-1 is a novel target for RNA binding protein CUGBP2

CUGBP2, a translation inhibitor, induces colon cancer cells to undergo apoptosis. Mcl-1, an antiapoptotic Bcl-2 family protein, interferes with mitochondrial activation to inhibit apoptosis. Here, we have determined the effect of CUGBP2 on Mcl-1 expression. We developed a HCUG2 cell line by stably expressing CUGBP2 in the HCT-116 colon cancer cells. HCUG2 cells demonstrate decreased levels of proliferation and increased apoptosis, compared with HCT-116 cells. Flow cytometry analysis demonstrated higher levels of cells in the G(2)-M phase. Western blot analyses demonstrated that there was decreased Bcl-2 and Mcl-1 protein but increased expression of Bax, cyclin B1, and Cdc2. Immunocytochemistry also demonstrated increased levels of cyclin B1 and Cdc2 in the nucleus of HCUG2 cells. However, there was colocalization of phosphorylated histone H3 with transferase-mediated dUTP nick-end labeling (TUNEL). Furthermore, immunostaining for alpha-tubulin demonstrated that there was disorganization of microtubules. These data suggest that CUGBP2 expression in HCUG2 cells induces the cells to undergo apoptosis during the G(2)-M phase of the cell cycle. We next determined the mechanism of CUGBP2-mediated reduction in Mcl-1 expression. Mcl-1 protein, but not Mcl-1 mRNA, was lower in HCUG2 cells, suggesting translation inhibition. CUGBP2 binds to Mcl-1 3'-untranslated region (3'-UTR) both in vitro and in HCUG2 cells. Furthermore, CUGBP2 increased the stability of both endogenous Mcl-1 and luciferase mRNA containing the Mcl-1 3'-UTR. However, luciferase protein expression from the luciferase-Mcl-1 3'-UTR mRNA was suppressed. Taken together, these data demonstrate that CUGBP2 inhibits Mcl-1 expression by inhibiting Mcl-1 mRNA translation, resulting in driving the cells to apoptosis during the G(2) phase of the cell cycle.     

Peripheral blood aspirates overexpressing IGF‐I via rAAV gene transfer undergo enhanced chondrogenic differentiation processes

Implantation of peripheral blood aspirates induced towards chondrogenic differentiation upon genetic modification in sites of articular cartilage injury may represent a powerful strategy to enhance cartilage repair. Such a single‐step approach may be less invasive than procedures based on the use of isolated or concentrated MSCs, simplifying translational protocols in patients. In this study, we provide evidence showing the feasibility of overexpressing the mitogenic and pro‐anabolic insulin‐like growth factor I (IGF‐I) in human peripheral blood aspirates via rAAV‐mediated gene transfer, leading to enhanced proliferative and chondrogenic differentiation (proteoglycans, type‐II collagen, SOX9) activities in the samples relative to control (reporter rAAV‐lacZ) treatment over extended periods of time (at least 21 days, the longest time‐point evaluated). Interestingly, IGF‐I gene transfer also triggered hypertrophic, osteo‐ and adipogenic differentiation processes in the aspirates, suggesting that careful regulation of IGF‐I expression may be necessary to contain these events in vivo. Still, the current results demonstrate the potential of targeting human peripheral blood aspirates via therapeutic rAAV transduction as a novel, convenient tool to treat articular cartilage injuries.     

Insights into the mechanism of a novel shockwave-assisted needle-free drug delivery device driven by in situ-generated oxyhydrogen mixture which provides efficient protection against mycobacterial infections

Background

Needle-free, painless and localized drug delivery has been a coveted technology in the area of biomedical research. We present an innovative way of trans-dermal vaccine delivery using a miniature detonation-driven shock tube device. This device utilizes~2.5 bar of in situ generated oxyhydrogen mixture to produce a strong shockwave that accelerates liquid jets to velocities of about 94 m/s.

Method

Oxyhydrogen driven shock tube was optimized for efficiently delivering vaccines in the intradermal region in vivo. Efficiency of vaccination was evaluated by pathogen challenge and host immune response. Expression levels of molecular markers were checked by qRT-PCR.

Results

High efficiency vaccination was achieved using the device. Post pathogen challenge with Mycobacterium tuberculosis, 100% survival was observed in vaccinated animals. Immune response to vaccination was significantly higher in the animals vaccinated using the device as compared to conventional route of vaccination.

Conclusion

A novel device was developed and optimized for intra dermal vaccine delivery in murine model. Conventional as well in-house developed vaccine strains were used to test the system. It was found that the vaccine delivery and immune response was at par with the conventional routes of vaccination. Thus, the device reported can be used for delivering live attenuated vaccines in the future.

     

Modulation of dendritic cell maturation and function by B lymphocytes

Investigating the signals that regulate the function of dendritic cells (DC), the sentinels of the immune system, is critical to understanding the role of DC in the regulation of immune responses. Accumulating lines of evidence indicate that in addition to innate stimuli and T cell-derived signals, B lymphocytes exert a profound regulatory effect in vitro and in vivo on the Ag-presenting function of DC. The identification of B cells as a cellular source of cytokines, chemokines, and autoantibodies that are critically involved in the process of maturation, migration, and function of DC provides a rationale for immunotherapeutic intervention of autoimmune and inflammatory conditions by targeting B cells. Conversely, efficient cross-presentation of Ags by DC pulsed with immune complexes provides an alternative approach in the immunotherapy of cancer and infectious diseases.     

Inhibition of bacterial RNase P by aminoglycoside-arginine conjugates

The potential of RNAs and RNA-protein (RNP) complexes as drug targets is currently being explored in various investigations. For example, a hexa-arginine derivative of neomycin (NeoR) and a tri-arginine derivative of gentamicin (R3G) were recently shown to disrupt essential RNP interactions between the trans-activator protein (Tat) and the Tat-responsive RNA (trans-activating region) in the human immunodeficiency virus (HIV) and also inhibit HIV replication in cell culture. Based on certain structural similarities, we postulated that NeoR and R3G might also be effective in disrupting RNP interactions and thereby inhibiting bacterial RNase P, an essential RNP complex involved in tRNA maturation. Our results indicate that indeed both NeoR and R3G inhibit RNase P activity from evolutionarily divergent pathogenic bacteria and do so more effectively than they inhibit partially purified human RNase P activity.     

Copper(II) complexes of new pentadentate bis(benzimidazolyl)-dithioether ligands: synthesis, structure, spectra and redox properties

Copper(II) complexes of a series of linear pentadentate ligands containing two benzimidazoles, two thioether sulfurs and a amine nitrogen, viz. N,N^′-bis{4^′-(2″-benzimidazolyl)(methyl)-3^′-thiabutyl}amine(L¹), N,N^′-bis{4^′-(2″-benzimidazolyl)(methyl)-3^′-thiabutyl}N-methylamine (L²), 2,6-bis{4^′-(2″-benzimidazolyl)(methyl)-3^′-thiabutyl}pyridine(L³), N,N^′-bis{4^′-(2″-benzimidazolyl)-2^′-thiabutyl}amine (L⁴), N,N^′-bis{4^′-(2″-benzimidazolyl)-2^′-thiabutyl}N-methylamine (L⁵) and 2,6-bis{4^′-(2″-benzimidazolyl)-2^′-thiabutyl}-3pyridine (L⁶) have been isolated and characterized by electronic absorption and EPR spectroscopy and cyclic and differential pulse voltammetry. Of these complexes, [Cu(L¹)](BF₄)₂ (1) and [Cu(L²)](BF₄)₂ (4) have been structurally characterized by X-ray crystallography. The coordination geometries around copper(II) in 1 and 4 are described as trigonal bipyramidal distorted square based pyramidal geometry (TBDSBP). The distorted CuN₃S basal plane in them is comprised of amine nitrogen, one thioether sulphur and two benzimidazole nitrogens and the other thioether sulfur is axially coordinated. The ligand field spectra of all the complexes are consistent with a mostly square-based geometry in solution. The EPR spectra of complexes [Cu(L¹)](BF₄)₂ (1), [Cu(L¹)](NO₃)₂ (2), [Cu(L²)](BF₄)₂ (4) and [Cu(L³)](ClO₄)₂ (6) are consistent with two species indicating the dissociation/disproportionation of the complex species in solution. All the complexes exhibit an intense CT band in the range 305-395 nm and show a quasireversible to irreversible Cu^II/Cu^I redox process with relatively positive E_1/2 values, which are consistent with the presence of two-coordinated thioether groups. The addition of N-methylimidazole (mim) replaces the coordinated thioether ligands in solution, as revealed from the negative shift (222-403 mV) in the Cu^II/Cu^I redox potential. The present study reveals that the effect of incorporating an amine nitrogen donor into CuN₂S₂ complexes is to generate an axial copper(II)-thioether coordination and also to enforce lesser trigonality on the copper(II) coordination geometry.     

Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.

The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.     

1,2,3,4-tetrahydrocarbazoles as 5-HT6 serotonin receptor ligands

An investigation of the structure-affinity relationships for the binding of 4-(N,N-dimethylaminomethyl)-N(9)-arylsulfonyl-9H-1,2,3,4-tetrahydrocarbazoles (conformationally-constrained analogues of the benzenesulfonyltryptamine 5-HT(6) antagonist MS-245) at human 5-HT(6) receptors revealed that various arylsulfonyl substituents are tolerated and that the 4-(N,N-dimethylaminomethyl) group is not required for binding. In particular, N(9)-(4-aminobenzenesulfonyl)-9H-1,2,3,4-tetrahydrocarbazole (20, K(i)=29 nM) was found to bind with high affinity and represents the first member of a new structural class of agents with 5-HT(6) antagonist properties (pA(2)=7.0; cAMP hydrolysis assay).