Acyl-lipid thioesterase1–4 from Arabidopsis thaliana form a novel family of fatty acyl–acyl carrier protein thioesterases with divergent expression patterns and substrate specificities

Hydrolysis of fatty acyl thioester bonds by thioesterases to produce free fatty acids is important for dictating the diversity of lipid metabolites produced in plants. We have characterized a four-member family of fatty acyl thioesterases from Arabidopsis thaliana, which we have called acyl-lipid thioesterase1 (ALT1), ALT2, ALT3, and ALT4. The ALTs belong to the Hotdog fold superfamily of thioesterases. ALT-like genes are present in diverse plant taxa, including dicots, monocots, lycophytes, and microalgae. The four Arabidopsis ALT genes were found to have distinct gene expression profiles with respect to each other. ALT1 was expressed specifically in stem epidermal cells and flower petals. ALT2 was expressed specifically in root endodermal and peridermal cells as well as in stem lateral organ boundary cells. ALT3 was ubiquitously expressed in aerial and root tissues and at much higher levels than the other ALTs. ALT4 expression was restricted to anthers. All four proteins were localized in plastids via an N-terminal targeting sequence of about 48 amino acids. When expressed in Escherichia coli, the ALT proteins used endogenous fatty acyl–acyl carrier protein substrates to generate fatty acids that varied in chain length (C6–C18), degree of saturation (saturated and monounsaturated), and oxidation state (fully reduced and β-ketofatty acids). Despite their high amino acid sequence identities, each enzyme produced a different profile of lipids in E. coli. The biological roles of these proteins are unknown, but they potentially generate volatile lipid metabolites that have previously not been reported in Arabidopsis.     

Thrombin-like serine protease,antiquorin from Euphorbia antiquorum latex induces platelet aggregation via PAR1-Akt/p38 signaling axis

Plant latex proteases (PLPs) are pharmacologically essential and are integral components of traditional medicine in the management of bleeding wounds. PLPs are known to promote blood coagulation and stop bleeding by interfering at various stages of hemostasis. There are a handful of scientific reports on thrombin-like enzymes characterized from plant latices. However, the role of plant latex thrombin-like enzymes in platelet aggregation is not well known. In the present study, we attempted to purify and characterize thrombin-like protease responsible for platelet aggregation. Among tested plant latices, Euphorbia genus latex protease fractions (LPFs) induced platelet aggregation. In Euphorbia genus, E. antiquorum LPF (EaLPF) strongly induced platelet aggregation and attenuated bleeding in mice. The purified thrombin-like serine protease, antiquorin (Aqn) is a glycoprotein with platelet aggregating activities that interfere in intrinsic and common pathways of blood coagulation cascade and alleviates bleeding and enhanced excision wound healing in mice. In continuation, the pharmacological inhibitor of PAR1 inhibited Aqn-induced phosphorylation of cPLA₂, Akt, and P38 in human platelets. Moreover, Aqn-induced platelet aggregation was inhibited by pharmacological inhibitors of PAR1, PI3K, and P38. These data indicate that PAR1-Akt/P38 signaling pathways are involved in Aqn-induced platelet aggregation. The findings of the present study may open up a new avenue for exploiting Aqn in the treatment of bleeding wounds.     

PEGylated rosin derivatives: Novel microencapsulating materials for sustained drug delivery

The aim of this study was to investigate PEGylated rosin derivatives (PRDs) as microencapsulating materials for sustained drug delivery. PRDs (D1, D2, and D3) composed of a constant weight of rosin and varied amounts of polyethylene glycol (PEG) 400 and maleic anhydride were synthesized in the laboratory. Microparticles were prepared by the O/O solvent evaporation technique using the acetone/paraffin system. Diclofenac sodium (DFS) and diltiazem hydrochloride (DLTZ) were used as model drugs. The effect of the type of PRD, drug, PRD:drug ratio, viscosity of external phase, stirring speed, concentration of magnesium stearate (droplet stabilizer), and method of preparation on particle size, drug loading, and drug release profiles of microparticles was investigated. PRDs could produce discrete and spherical microspheres (with DFS) and microcapsules (with DLTZ). The drug loading value for microparticles was found to be in the range of 37.21% to 87.90%. The microparticle size range was 14 to 36 μm. The particle size and drug loadings of microparticles were substantially affected by the concentration of magnesium stearate and the type of drug, respectively. Most of the formulations could sustain the DFS and DLTZ release for 20 hours. DFS and DLTZ release from PRD microparticles followed Hixson-Crowell and first-order kinetics, respectively. The results suggest that PRDs can be used successfully to prepare discrete and spherical microparticles with DFS and DLTZ for sustained drug delivery. Published: June 22, 2007     

Investigating the Causes for Decreased Levels of Glutathione in Individuals with Type II Diabetes

Tuberculosis (TB) remains an eminent global burden with one third of the world’s population latently infected with Mycobacterium tuberculosis (M. tb). Individuals with compromised immune systems are especially vulnerable to M. tb infection. In fact, individuals with Type 2 Diabetes Mellitus (T2DM) are two to three times more susceptible to TB than those without T2DM. In this study, we report that individuals with T2DM have lower levels of glutathione (GSH) due to compromised levels of GSH synthesis and metabolism enzymes. Transforming growth factor beta (TGF-β), a cytokine that is known to decrease the expression of the catalytic subunit of glutamine-cysteine ligase (GCLC) was found in increased levels in the plasma samples from individuals with T2DM, explaining the possible underlying mechanism that is responsible for decreased levels of GSH in individuals with T2DM. Moreover, increased levels of pro-inflammatory cytokines such as interleukin-6 (IL-6) and interleukin-17 (IL-17) were observed in plasma samples isolated from individuals with T2DM. Increased levels of IL-6 and IL-17 was accompanied by enhanced production of free radicals further indicating an alternative mechanism for the decreased levels of GSH in individuals with T2DM. Augmenting the levels of GSH in macrophages isolated from individuals with T2DM resulted in improved control of M. tb infection. Furthermore, cytokines that are responsible for controlling M. tb infection at the cellular and granuloma level such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interferon-gamma (IFN-γ), and interleukin-12 (IL-12), were found to be compromised in plasma samples isolated from individuals with T2DM. On the other hand, interleukin-10 (IL-10), an immunosuppressive cytokine was increased in plasma samples isolated from individuals with T2DM. Overall, these findings suggest that lower levels of GSH in individuals with T2DM lead to their increased susceptibility to M. tb infection.     

Application of optical imaging and spectroscopy to radiation biology

Optical imaging and spectroscopy is a diverse field that has been of critical importance in a wide range of areas in radiation research. It is capable of spanning a wide range of spatial and temporal scales, and has the sensitivity and specificity needed for molecular and functional imaging. This review will describe the basic principles of optical imaging and spectroscopy, highlighting a few relevant applications to radiation research.     

Golgi Phosphoprotein 3 Determines Cell Binding Properties under Dynamic Flow by Controlling Golgi Localization of Core 2 N-Acetylglucosaminyltransferase 1

Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLe^x), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR⁹ sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT^1–16-Leu^17–32-Gly^33–42-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA⁹) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLe^x associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.