DMT1: which metals does it transport?

DMT1-Divalent Metal (Ion) Transporter 1 or SLC11A2/DCT1/Nramp2 - transports Fe2+ into the duodenum and out of the endosome during the transferrin cycle. DMTI also is important in non-transferrin bound iron uptake. It plays similar roles in Mn2+ trafficking. Voltage clamping showed that six other metals evoked currents, but it is unclear if these metals are substrates for DMT1. This report summarizes progress on which metals DMT1 transports, focusing on results from the authors' labs. We recently cloned 1A/+IRE and 2/-IRE DMT1 isoforms to generate HEK293 cell lines that express them in a tetracycline-inducible fashion, then compared induced expression to uninduced expression and to endogenous DMT1 expression. Induced expression increases approximately 50x over endogenous expression and approximately 10x over uninduced levels. Fe2+, Mn2+, Ni2+ and Cu1+ or Cu2+ are transported. We also explored competition between metal ions using this system because incorporation essentially represents DMT1 transport and find this order for transport affinity: Mn>?Cd>?Fe>Pb-Co-Ni>Zn. The effects of decreased DMT1 also could be examined. The Belgrade rat has diminished DMT1 function and thus provides ways of testing. A series of DNA constructs that generate siRNAs specific for DMT1 or certain DMT1 isoforms yield another way to test DMT1-based transport.     

Dicer-2-Dependent Activation of Culex Vago Occurs via the TRAF-Rel2 Signaling Pathway

Despite their importance as vectors of human and livestock diseases, relatively little is known about innate antiviral immune pathways in mosquitoes and other insects. Previous work has shown that Culex Vago (CxVago), which is induced and secreted from West Nile virus (WNV)-infected mosquito cells, acts as a functional homolog of interferon, by activating Jak-STAT pathway and limiting virus replication in neighbouring cells. Here we describe the Dicer-2-dependent pathway leading to WNV-induced CxVago activation. Using a luciferase reporter assay, we show that a NF-κB-like binding site in CxVago promoter region is conserved in mosquito species and is responsible for induction of CxVago expression following WNV infection. Using dsRNA-based gene knockdown, we show that the NF-κB ortholog, Rel2, plays significant role in the signaling pathway that activates CxVago in mosquito cells in vitro and in vivo. Using similar approaches, we also show that TRAF, but not TRAF-3, is involved in activation of Rel2 after viral infection. Overall the study shows that a conserved signaling pathway, which is similar to mammalian interferon activation pathway, is responsible for the induction and antiviral activity of CxVago.     

Design and evaluation of deformable talc agglomerates prepared by crystallo-co-agglomeration technique for generating heterogeneous matrix

The crystallo-co-agglomeration technique was used to design directly compressible and deformable agglomerates of talc containing the low-dose drug bromhexine hydrochloride (BXH). The process of agglomeration involved the use of dichloromethane as a good solvent and bridging liquid for BXH, water as a poor solvent, talc as diluent, and Tween 80 to aid dispersion of BXH and diluent into the poor solvent. Hydroxypropyl methylcellulose (50 cps) 4% wt/wt was used to impart the desired mechanical strength and polyethylene glycol 6000 5% wt/wt was used to impart the desired sphericity to the agglomerates. Clarity of the supernatant was considered an endpoint for completion of the agglomeration process. The drug-to-talc ratio in optimized batch 1 (BT1) and batch 2 (BT2) was kept at 1:15.66 and 1:24, respectively. The spherical agglomerates obtained were evaluated for topographic, micromeritic, mechanical, deformation, compressional, and drug release properties. The agglomeration yield and drug entrapment for both batches were above 94% wt/wt. Crushing strength and friability studies showed good handling qualities of agglomerates. Heckel plot studies showed low mean yield pressure and high tensile strength, indicating excellent compressibility and compactibility of agglomerates. Diametral and axial fracture of compacts showed deformation of agglomerates revealing formation of a heterogeneous compact. Drug release was sustained for 9 hours and 5 hours from BT1 and BT2, respectively, in 0.1N HCl. Hence, the crystallo-co-agglomeration technique can be successfully used for obtaining spherical, deformable, and directly compressible agglomerates, generating a heterogeneous matrix system and providing sustained drug release.     

Exploring Microstructural Changes in Structural Analogues of Ibuprofen-Hosted In Situ Gelling System and Its Influence on Pharmaceutical Performance

The present work explores inner structuration of in situ gelling system consisting of glyceryl monooleate (GMO) and oleic acid (OA). The system under study involves investigation of microstructural changes which are believed to govern the pharmaceutical performance of final formulation. The changes which are often termed mesophasic transformation were analysed by small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), rheology and plane polarised light (PPL) microscopy. The current work revealed transformation of blank system from W/O emulsion to reverse hexagonal structure upon addition of structural analogues of ibuprofen. Such transformations are believed to occur due to increased hydrophobic volume within system as probed by SAXS analysis. The findings of SAXS studies were well supported by DSC, rheology and PPL microscopy. The study established inverse relationship between log P value of structural analogues of ibuprofen and the degree of binding of water molecules to surfactant chains. Such relationship had pronounced effect on sol–gel transformation process. The prepared in situ gelling system showed sustained drug release which followed Higuchi model.KEY WORDS: flurbiprofen, hexagonal phase, ibuprofen, ketoprofen, liquid crystal, sustained drug release     

Studies on spray-dried mixtures of chitosan and hydrolyzed gelatin as tablet binder: A technical note

Conclusion  The results of this investigation showed that spray drying of CH and HG resulted in free-flowing powder, which exhibited improved compressibility and compactibility, thereby suggesting the potential of such spray-dried mixtures of CH and HG as binder in tablets. Optimization of CH:HG ratio needs to be undertaken on a case basis, depending on the presence of other excipients in the formulation. Published: October 22, 2005     

Decoupling ferritin synthesis from free cytosolic iron results in ferritin secretion

Ferritin is a multisubunit protein that is responsible for storing and detoxifying cytosolic iron. Ferritin can be found in serum but is relatively iron poor. Serum ferritin occurs in iron overload disorders, in inflammation, and in the genetic disorder hyperferritinemia with cataracts. We show that ferritin secretion results when cellular ferritin synthesis occurs in the relative absence of free cytosolic iron. In yeast and mammalian cells, newly synthesized ferritin monomers can be translocated into the endoplasmic reticulum and transits through the secretory apparatus. Ferritin chains can be translocated into the endoplasmic reticulum in an in?vitro translation and membrane insertion system. The insertion of ferritin monomers into the ER occurs under low-free-iron conditions, as iron will induce the assembly of ferritin. Secretion of ferritin chains provides a mechanism that limits ferritin nanocage assembly and ferritin-mediated iron sequestration in the absence of the translational inhibition of ferritin synthesis.     

Agglomeration of ibuprofen with talc by novel crystallo-co-agglomeration technique

The purpose of this research work was to obtain directly compressible agglomerates of ibuprofen with talc by a novel crystallo-co-agglomeration (CCA) technique, which is an extension of spherical crystallization. Ibuprofen-talc agglomerates were prepared using dichloromethane (DCM)-water as the crystallization system. DCM acted as a good solvent for ibuprofen as well as a bridging liquid for agglomeration of crystallized drug with talc. The agglomerates were characterized by differential scanning calorimetry, powder X-ray diffraction, and scanning electron microscopy and were evaluated for tableting properties and for drug release. The process yielded spherical agglomerates containing ∼95% to 96% wt/wt of ibuprofen. Agglomerates containing talc showed uniform distribution of hydroxypropylmethylcellulose and decreased crystallinity, and deformed under pressure. The miniscular form of ibuprofen and the hydrophobicity of talc governed the drug release rate. The batch containing a higher proportion of talc showed zeroorder kinetics and drug release was extended up to 13 hours. The CCA technique developed in this study is suitable for obtaining agglomerates of drug with talc as an excipient.     

Virology at the Lorne Infection and Immunity Conference 2019

<正>The Lorne Infection and Immunity Conference is one of five scientific meetings held during each month of February at the Cumberland resort in the picturesque seaside town of Lorne, on the Great Ocean Road in Victoria (Australia).The specific aim of the meeting is to bring together basic,