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Salojin K.V.,Lexicon Pharmaceuticals Inc. | Cabrera R.M.,Texas A&M University | Cabrera R.M.,University of Texas at Austin | Sun W.,Lexicon Pharmaceuticals Inc. | And 11 more authors.
Blood | Year: 2011

The human proton coupled folate transporter (PCFT) is involved in low pH-dependent intestinal folate transport. In this report, we describe a new murine model of the hereditary folate malabsorption syndrome that we developed through targeted disruption of the first 3 coding exons of the murine homolog of the PCFT gene. By 4 weeks of age, PCFT-deficient (PCFT-/-) mice developed severe macrocytic normochromic anemia and pancytopenia. Immature erythroblasts accumulated in the bone marrow and spleen of PCFT-/- mice and failed to differentiate further, showing an increased rate of apoptosis in intermediate erythroblasts and reduced release of reticulocytes. In response to the inefficient hematologic development, the serum of the PCFT-/- animals contained elevated concentrations of erythropoietin, soluble transferrin receptor (sCD71), and thrombopoietin. In vivo folate uptake experiments demonstrated a systemic folate deficiency caused by disruption of PCFT-mediated intestinal folate uptake, thus confirming in vivo a critical and nonredundant role of the PCFT protein in intestinal folate transport and erythropoiesis. The PCFT-deficient mouse serves as a model for the hereditary folate malabsorption syndrome and is the most accurate animal model of folate deficiency anemia described to date that closely captures the spectrum of pathology typical of this disease. © 2011 by The American Society of Hematology.

Moussa S.H.,Texas A&M University | Kuznetsov V.,Texas A&M University | Tran T.A.T.,University of Texas Southwestern Medical Center | Sacchettini J.C.,Texas A&M University | And 2 more authors.
Protein Science | Year: 2012

Genetic studies have established that lysis inhibition in bacteriophage T4 infections occurs when the RI antiholin inhibits the lethal hole-forming function of the T holin. The T-holin is composed of a single N-terminal transmembrane domain and a ∼20 kDa periplasmic domain. It accumulates harmlessly throughout the bacteriophage infection cycle until suddenly causing permeabilization of the inner membrane, thereby initiating lysis. The RI antiholin has a SAR domain that directs its secretion to the periplasm, where it can either be inactivated and degraded or be activated as a specific inhibitor of T. Previously, it was shown that the interaction of the soluble domains of these two proteins within the periplasm was necessary for lysis inhibition. We have purified and characterized the periplasmic domains of both T and RI. Both proteins were purified in a modified host that allows disulfide bond formation in the cytoplasm, due to the functional requirement of conserved disulfide bonds. Analytical centrifugation and circular dichroism spectroscopy showed that RI was monomeric and exhibited ∼80% alpha-helical content. In contrast, T exhibited a propensity to oligomerize and precipitate at high concentrations. Incubation of RI with T inhibits this aggregation and results in a complex of equimolar T and RI content. Although gel filtration analysis indicated a complex mass of 45 kDa, intermediate between the predicted 30 kDa heterodimer and 60 kDa heterotetramer, sedimentation velocity analysis indicated that the predominant species is the former. These results suggest that RI binding to T is necessary and sufficient for lysis inhibition. © 2012 The Protein Society.

Arockiasamy A.,Texas A&M University | Aggarwal A.,Texas A&M University | Savva C.G.,Texas A&M University | Holzenburg A.,Texas A&M University | And 2 more authors.
Protein Science | Year: 2011

In eukaryotes, calcium-binding proteins play a pivotal role in diverse cellular processes, and recent findings suggest similar roles for bacterial proteins at different stages in their life cycle. Here, we report the crystal structure of calcium dodecin, Rv0379, from Mycobacterium tuberculosis with a dodecameric oligomeric assembly and a unique calcium-binding motif. Structure and sequence analysis were used to identify orthologs of Rv0379 with different ligand-binding specificity. © 2011 The Protein Society.

Ichite N.,Florida A&M University | Chougule M.,University of Hawaii at Hilo | Patel A.R.,Florida A&M University | Jackson T.,Florida A&M University | And 3 more authors.
Molecular Cancer Therapeutics | Year: 2010

The purpose of this study was to determine the anticancer efficacy of 1,1-bis (3′-indolyl)-1-(p-biphenyl) methane (DIM-C-pPhC6H 5) by inhalation delivery alone and in combination with i.v. docetaxel in a murine model for lung cancer. An aqueous DIM-C-pPhC 6H5 formulation was characterized for its aerodynamic properties. Tumor-bearing athymic nude mice were exposed to nebulized DIM-C-pPhC6H5, docetaxel, or combination (DIM-C-pPhC 6H5 plus docetaxel) using a nose-only exposure technique. The aerodynamic properties included mass median aerodynamic diameter of 1.8 ± 0.3 μm and geometric SD of 2.31 ± 0.02. Lung weight reduction in mice treated with the drug combination was 64% compared with 40% and 47% in mice treated with DIM-C-pPhC6H5 aerosol and docetaxel alone, respectively. Combination treatment decreased expression of Akt, cyclin D1, survivin, Mcl-1, NF-κB, IκBα, phospho-IκBα, and vascular endothelial growth factor (VEGF) and increased expression of c-Jun NH2-terminal kinase 2 and Bad compared with tumors collected from single-agent treatment and control groups. DNA fragmentation was also enhanced in mice treated with the drug combination compared with docetaxel or DIM-C-pPhC6H5 alone. Combination treatment decreased expressions of VEGF and CD31 compared with single-agent treated and control groups. These results suggest that DIM-C-pPhC6H5 aerosol enhanced the anticancer activity of docetaxel in a lung cancer model by activating multiple signaling pathways. The study provides evidence that DIM-C-pPhC6H5 can be used alone or in combination with other drugs for the treatment of lung cancer using the inhalation delivery approach. ©2010 AACR.

Humtsoe J.O.,University of California at San Francisco | Liu M.,Institute of Biosciences and Technology | Malik A.B.,University of Illinois at Chicago | Wary K.K.,University of Illinois at Chicago
Molecular and Cellular Biology | Year: 2010

Endothelial cell (EC) migration, cell-cell adhesion, and the formation of branching point structures are considered hallmarks of angiogenesis; however, the underlying mechanisms of these processes are not well understood. Lipid phosphate phosphatase 3 (LPP3) is a recently described p120-catenin-associated integrin ligand localized in adherens junctions (AJs) of ECs. Here, we tested the hypothesis that LPP3 stimulates β-catenin/lymphoid enhancer binding factor 1 (β-catenin/LEF-1) to induce EC migration and formation of branching point structures. In subconfluent ECs, LPP3 induced expression of fibronectin via β-catenin/LEF-1 signaling in a phosphatase and tensin homologue (PTEN)-dependent manner. In confluent ECs, depletion of p120-catenin restored LPP3-mediated β-catenin/LEF-1 signaling. Depletion of LPP3 resulted in destabilization of β-catenin, which in turn reduced fibronectin synthesis and deposition, which resulted in inhibition of EC migration. Accordingly, reexpression of β-catenin but not p120-catenin in LPP3-depleted ECs restored de novo synthesis of fibronectin, which mediated EC migration and formation of branching point structures. In confluent ECs, however, a fraction of p120-catenin associated and colocalized with LPP3 at the plasma membrane, via the C-terminal cytoplasmic domain, thereby limiting the ability of LPP3 to stimulate β-catenin/LEF-1 signaling. Thus, our study identified a key role for LPP3 in orchestrating PTEN-mediated β-catenin/LEF-1 signaling in EC migration, cell-cell adhesion, and formation of branching point structures. Copyright © 2010, American Society for Microbiology.

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