Otsuka Maryland Medicinal Laboratories Inc.

Dunkirk Town Center, MD, United States

Otsuka Maryland Medicinal Laboratories Inc.

Dunkirk Town Center, MD, United States

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Rai U.,Rockefeller University | Huang J.,Rockefeller University | Mishra S.,New York University | Li X.,Rockefeller University | And 3 more authors.
Biomolecules | Year: 2012

Hydrodynamic tail vein (HTV) delivery is a simple and rapid tail vein injection method of a high volume of naked plasmid DNA resulting in high levels of foreign gene expression in organs, especially the liver. Compared to other organs, HTV delivery results in more than a 1000-fold higher transgene expression in liver. After being bitten by malaria-infected mosquitoes, malaria parasites transiently infect the host liver and form the liver stages. The liver stages are known to be the key target for CD8+ T cells that mediate protective anti-malaria immunity in an animal model. Therefore, in this study, we utilized the HTV delivery technique as a tool to determine the in vivo cytotoxic effect of malaria antigen-specific CD8+ T cells. Two weeks after mice were immunized with recombinant adenoviruses expressing malarial antigens, the immunized mice as well as naïve mice were challenged by HTV delivery of naked plasmid DNA co-encoding respective antigen together with luciferase using dual promoters. Three days after the HTV challenge, non-invasive whole-body bioluminescent imaging was performed. The images demonstrate in vivo activity of CD8+ T cells against malaria antigen-expressing cells in liver. © 2012 by the authors; licensee MDPI, Basel, Switzerland.


Kawashima Y.,U.S. National Institutes of Health | Kawashima Y.,Tsuchiura Kyodo General Hospital | Geleoc G.S.G.,University of Virginia | Geleoc G.S.G.,Harvard University | And 14 more authors.
Journal of Clinical Investigation | Year: 2011

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1Δ mice) were deaf and those with a deletion of Tmc2 (Tmc2Δ mice) were phenotypically normal, Tmc1ΔTmc2Δ mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1ΔTmc2Δ mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.


Zhang Y.,Otsuka Maryland Medicinal Laboratories Inc. | Tian D.,University of Minnesota | Matsuyama H.,Otsuka Maryland Medicinal Laboratories Inc. | Hamazaki T.,Osaka City University | And 6 more authors.
Journal of Biomolecular Screening | Year: 2016

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast "knock-out/knock-in" screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival. © 2015 Society for Laboratory.


PubMed | University of Minnesota, Florida College, Otsuka Maryland Medicinal Laboratories Inc. and Osaka City University
Type: Journal Article | Journal: Journal of biomolecular screening | Year: 2016

Transport of ADP and ATP across mitochondria is one of the primary points of regulation to maintain cellular energy homeostasis. This process is mainly mediated by adenine nucleotide translocase (ANT) located on the mitochondrial inner membrane. There are four human ANT isoforms, each having a unique tissue-specific expression pattern and biological function, highlighting their potential as drug targets for diverse clinical indications, including male contraception and cancer. In this study, we present a novel yeast-based high-throughput screening (HTS) strategy to identify compounds inhibiting the function of ANT. Yeast strains generated by deletion of endogenous proteins with ANT activity followed by insertion of individual human ANT isoforms are sensitive to cell-permeable ANT inhibitors, which reduce proliferation. Screening hits identified in the yeast proliferation assay were characterized in ADP/ATP exchange assays employing recombinant ANT isoforms expressed in isolated yeast mitochondria and Lactococcus lactis as well as by oxygen consumption rate in mammalian cells. Using this approach, closantel and CD437 were identified as broad-spectrum ANT inhibitors, whereas leelamine was found to be a modulator of ANT function. This yeast knock-out/knock-in screening strategy is applicable to a broad range of essential molecular targets that are required for yeast survival.


Suzuki H.I.,University of Tokyo | Suzuki H.I.,Massachusetts Institute of Technology | Katsura A.,University of Tokyo | Matsuyama H.,University of Tokyo | And 2 more authors.
Oncogene | Year: 2014

Cancer initiation and progression are defined by the behavior of cancer cells per se and the development of tumor tissues, both of which are modulated by crosstalk between cancer cells and the surrounding microenvironment. Advances in cancer research have highlighted the significance of constant evolution of the tumor microenvironment, leading to tumor formation, metastasis and refractoriness to therapy. MicroRNAs (miRNAs) are small non-coding RNAs that function as major players of posttranscriptional gene regulation in diverse biological processes. They function as both tumor suppressors and promoters in many aspects of the autonomous behavior of cancer cells. Theoretically, dysfunction in the gene regulatory networks of cancer cells is one of the major driving forces for alterations of ostensibly normal surrounding cells. In this context, the core targets of miRNAs, termed miRNA regulons, are currently being expanded to include various modulators of the tumor microenvironment. Recent advances have highlighted two important roles played by miRNAs in the evolution of tumor microenvironments: miRNAs in tumor cells transform the microenvironment via non-cell-autonomous mechanisms, and miRNAs in neighboring cells stabilize cancer hallmark traits. These observations epitomize the distal and proximal functions of miRNAs in tumor microenvironments, respectively. Such regulation by miRNAs affects tumor angiogenesis, immune invasion and tumor-stromal interactions. This review summarizes recent findings on the mechanisms of miRNA-mediated regulation of tumor microenvironments, with a perspective on the design of therapeutic interventions. © 2015 Macmillan Publishers Limited All rights reserved.


Shakur Y.,Otsuka Maryland Medicinal Laboratories Inc | De Koning H.P.,University of Glasgow | Ke H.,University of North Carolina at Chapel Hill | Kambayashi J.,Otsuka Maryland Medicinal Laboratories Inc | Seebeck T.,University of Bern
Handbook of Experimental Pharmacology | Year: 2011

Protozoan parasites of the order kinetoplastida are the causative agents of three of the world's most important neglected human diseases: African trypanosomiasis, American trypanosomiasis, and leishmaniasis. Current therapies are limited, with some treatments having serious and sometimes lethal side effects. The growing number of cases that are refractory to treatment is also of concern. With few new drugs in development, there is an unmet medical need for new, more effective, and safer medications. Recent studies employing genetic and pharmacological techniques have begun to shed light on the role of the cyclic nucleotide phosphodiesterases in the life cycle of these pathogens and suggest that these important regulators of cyclic nucleotide signaling may be promising new targets for the treatment of parasitic diseases. © 2011 Springer-Verlag Berlin Heidelberg.


Zhang J.,U.S. National Institutes of Health | Qiu S.,U.S. National Institutes of Health | Qiu S.,Dickinson College | Zhang Y.,U.S. National Institutes of Health | And 7 more authors.
Anticancer Research | Year: 2012

Background/Aim: To determine if early passage tumor cells obtained from patients with mesothelioma continue to express the tumor differentiation antigen mesothelin and their sensitivity to the anti-mesothelin immunotoxin SS1P. Materials and Methods: Cell cultures were established from ascites or pleural effusion of 6 peritoneal and 3 pleural mesothelioma patients, respectively. These cells were evaluated for mesothelin expression by immunohistochemistry and flow cytometry. Results: Although mesothelin was highly expressed in tumor biopsies of all patients, only 3 out of 9 malignant effusions from these patients when grown in short-term culture showed strong mesothelin positivity by IHC. By flow cytometry, the number of mesothelin sites per cell was variable ranging from 580 to 210,000 sites/cell. Cells with strong mesothelin expression by IHC and increased number of mesothelin sites/cell were sensitive to SS1P. Conclusions: Most mesothelioma tumors loose mesothelin when grown in vitro and the sensitivity of these cells to SS1P is dependent on the number of mesothelin sites/cell.


Yang X.-M.,University of South Alabama | Liu Y.,University of South Alabama | Liu Y.,Otsuka Maryland Medicinal Laboratories Inc. | Tandon N.,Otsuka Maryland Medicinal Laboratories Inc. | And 3 more authors.
Basic Research in Cardiology | Year: 2010

Ischemic pre- (IPC) and post- (IPOC) conditioning are very protective in laboratory animals, but it has not been possible to measure their anti-infarct potency in human hearts. Non-human primates are genetically closer to humans than other laboratory animals, but until now there have been no studies of IPC or IPOC in any primate species. Accordingly the left anterior descending coronary artery of cynomolgus monkeys was occluded for 90 min and reperfused for 4 h. In control animals, only 44% of the risk zone infarcted indicating cynomolgus myocardium is much more resistant to infarction than that of rabbits or rats. The regression line for the infarct-risk zone plot was very linear (r = 0.99), and intersected the risk zone axis at 0.82 cm3. Even small changes in infarct size could be detected as a shift in this line. Collateral flow in 12 monkeys was 6.6% of flow to normal myocardium and not a covariate of infarct size. IPC with two cycles of 10-min coronary occlusion/10-min reperfusion reduced infarction to near zero indicating that the innate resistance to infarction was not caused by constitutive preconditioning. Wortmannin, an antagonist of phosphatidylinositol 3-kinase (PI3-K), administered just before release of the 90-min coronary occlusion attenuated IPC's infarct-sparing effect by ∼50% suggesting that PI3-K was involved in preconditioning's protection. IPOC with six cycles of 30-s reperfusion/30-s coronary reocclusion, a very protective protocol in most species, was much less protective than IPC. We conclude that ischemic preconditioning is extremely protective in cynomolgus hearts despite their sparse collateralization but, surprisingly, the protocol of IPOC used in this study offers less protection. © 2009 Springer-Verlag.


Fong M.,Otsuka Maryland Medicinal Laboratories Inc. | Yoshitake M.,Otsuka Maryland Medicinal Laboratories Inc. | Kambayashi J.,Otsuka Maryland Medicinal Laboratories Inc. | Liu Y.,Otsuka Maryland Medicinal Laboratories Inc.
Circulation Journal | Year: 2010

Background: The mechanisms underlying the ability of cilostazol to improve walking distance in patients with intermittent claudication (IC) are not fully understood, but may be related to its phosphodiesterase type 3 (PDE3) and adenosine uptake inhibition. In the present study the effect of cilostazol on blood flow and interstitial adenosine concentration was compared with that of the PDE3 inhibitor, milrinone, and the adenosine uptake inhibitor, draflazine. Methods and Results: Rabbit gastrocnemius muscle blood flow was measured under resting, contracting and ischemic conditions. Interstitial adenosine was sampled by microdialysis. None of the drugs affected tissue blood flow at rest. Blood flow in electrically stimulated muscle was 2- to 3-fold higher in vehicle-, milrinone- and draflazine-treated animals. However, cilostazol caused an 8-fold increase. Ligation of the femoral artery decreased blood flow in the stimulated muscle in all groups to a similar degree. Cilostazol and draflazine increased the dialysate adenosine concentration during the first 10 min of muscle contraction, but had no effect during ischemia, most likely because of the high AMP deaminase activity in skeletal muscle. Conclusions: Cilostazol increases blood flow in the gastrocnemius muscle during contraction and it is this effect that may be partially responsible for the improved walking distance in IC patients.


PubMed | Otsuka Maryland Medicinal Laboratories Inc
Type: | Journal: Handbook of experimental pharmacology | Year: 2011

Protozoan parasites of the order kinetoplastida are the causative agents of three of the worlds most important neglected human diseases: African trypanosomiasis, American trypanosomiasis, and leishmaniasis. Current therapies are limited, with some treatments having serious and sometimes lethal side effects. The growing number of cases that are refractory to treatment is also of concern. With few new drugs in development, there is an unmet medical need for new, more effective, and safer medications. Recent studies employing genetic and pharmacological techniques have begun to shed light on the role of the cyclic nucleotide phosphodiesterases in the life cycle of these pathogens and suggest that these important regulators of cyclic nucleotide signaling may be promising new targets for the treatment of parasitic diseases.

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