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KALAMZOO, MI, United States

Graf M.,National University of Singapore | Graf M.,Proteos, Inc. | Qi-Wen E.-R.T.,National University of Singapore | Sarusie M.V.,National University of Singapore | And 2 more authors.
Molecular Endocrinology | Year: 2015

Dmrt transcription factors control sex determination or sex-specific differentiation across all invertebrate and vertebrate species, in which they have been studied so far. In addition to important functions in the reproductive system, also nongonadal roles have been assigned to several dmrt family members. One example is dmrt5, which was shown to guide neurogenesis in the forebrain of some vertebrates including fish. Here we show that in zebrafish, dmrt5 is also expressed adjacent to the pituitary anlage and later in the anterior pars distalis in which it organizes differentiation of endocrine cells. We find that pituitary induction, cell survival, proliferation, and early lineage specification in the pituitary is independent of dmrt5. Instead, dmrt5 is required for terminal differentiation of corticotropes and gonadotropes. Gene knockdown and mutant analysis revealed that dmrt5 promotes corticotrope differentiation via tbx19 expression, whereas it prevents gonadotrope differentiation in the anterior pars distalis. In dmrt5 morphants and mutants, reduced corticotrope numbers may result in irregular positioning and reduced maintenance of lactotropes. In conclusion, our study establishes a novel function for dmrt5 for cell differentiation in the anterior pituitary. Intriguingly, its effect on gonadotrope numbers defines a first nongonadal role for a dmrt family member that appears crucial for the activity of the reproductive system. © 2015 by the Endocrine Society.

Luu Y.,University of California at San Diego | Junker W.,University of Nebraska Medical Center | Rachagani S.,University of Nebraska Medical Center | Das S.,University of Nebraska Medical Center | And 4 more authors.
International Journal of Biochemistry and Cell Biology | Year: 2010

The membrane-bound mucins, MUC17 (human) and Muc3 (mouse), are highly expressed on the apical surface of intestinal epithelia and are thought to be cytoprotective. The extracellular regions of these mucins contain EGF-like Cys-rich segments (CRD1 and CRD2) connected by an intervening linker domain (L). The purpose of this study was to determine the functional activity of human MUC17 membrane-bound mucin. Methods: Endogenous MUC17 was inhibited in LS174T colon cells by stable transfection of a small hairpin RNA targeting MUC17 (LSsi cells). The effect of recombinant MUC17-CRD1-L-CRD2 protein on migration, apoptosis, and experimental colitis was determined. Results: Reduced MUC17 expression in LSsi cells was associated with visibly reduced cell aggregation, reduced cell-cell adherence, and reduced cell migration, but no change in tumorigenicity. LSsi cells also demonstrated a 3.7-fold increase in apoptosis rates compared with control cells following treatment with etoposide. Exposure of colonic cell lines to exogenous recombinant MUC17-CRD1-L-CRD2 protein significantly increased cell migration and inhibited apoptosis. As a marker of biologic activity, MUC17-CRD1-L-CRD2 proteins stimulate ERK phosphorylation in colonic cell lines; and inhibition of ERK phosphorylation reduced the anti-apoptosis and migratory effect of MUC17-CRD1-L-CRD2. Finally, mice treated with MUC17-CRD1-L-CRD2 protein given per rectum demonstrated accelerated healing in acetic acid and dextran sodium sulfate induced colitis in vivo. These data indicate that both native MUC17 and the exogenous recombinant cysteine-rich domain of MUC17 play a role in diverse cellular mechanisms related to cell restitution, and suggest a potential role for MUC17-CRD1-L-CRD2 recombinant protein in the treatment of mucosal inflammatory diseases. © 2010.

Sandin S.,Nanyang Technological University | Rhodes D.,Proteos, Inc.
Current Opinion in Structural Biology | Year: 2014

The telomerase reverse transcriptase has an essential role in telomere maintenance and in cancer biology. Progress during the last year has revealed the three-dimensional architecture of both human and ciliate telomerase at about 25. Å resolution, obtained using single particle electron microscopy (EM). The structural analysis of the two holoenzyme complexes isolated from cells shows that whilst the ciliate telomerase is monomeric, the human telomerase is dimeric and only functional as a dimer. We critically discuss the approaches taken to assign the location of protein and RNA subunits, as well as fitting the crystal structure of the catalytic protein subunit in the medium resolution EM density maps. Comparison of the two structural interpretations reveals not only a common RNA/reverse transcriptase core, but also significant differences due to different RNA subunit size and protein composition. These differences suggest that the oligomeric state and subunit composition of telomerase in evolutionary distant organism have evolved. © 2014.

Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 190.12K | Year: 2005

DESCRIPTION (provided by applicant): The goal of this Phase 1 proposal is to optimize the activity and minimize the size of a protein therapeutic for intestinal bowel diseases (IBD), in order to enable production of the protein in quantities needed for trials in humans. Currently, there is no safe and effective treatment for IBD. Professor Samuel Ho and colleagues at the University of Minnesota have produced a portion of a naturally occurring murine intestinal mucin known to be protective against IBD that is effective in a mouse model of colitis. This GST-tagged protein contains two small domains that are similar to epidermal growth factor (EGF) and that we refer to as EFD units, linked by a peptide segment i.e.: GST(279aa)-EFD1 (approximately 50aa) -Linker(approximately 120aa) -EFD2(approximately 60aa). Despite the similarity of these EFD units to EGF, the mucin-derived protein promotes wound healing by a mechanism that does not involve activation of the EGF receptor, therefore lowering its potential for carcinogenicity. There are problems with production of this large protein in quantities needed for clinical trials, and the specific aims of this proposal are to optimize the current candidate protein by exploration of constructs with different EFD units from human mucins, truncation of the linker segment, and removal of the GST tag. Trefoil factors are naturally occurring EFD units that are produced in the human intestine and that promote wound healing. These variants, together with the EFD units of human mucins MUC3 and MUC17, will be incorporated by cassette mutagenesis into the mucin structural format. With the objective of achieving a MW < 15,000, the linker segment will be truncated to the shortest length consistent with reservation of biological activity. Proteins lacking purification tags will be produced at Proteos by bacterial expression and if inclusion bodies are formed, Proteos scientists have been successful in refolding such proteins. Constructs will be assayed in cell-migration and proliferation assays, and in the mouse model of ulcerative colitis applied in the Ho laboratory at the University of Minnesota in earlier studies of the first generation murine mucin protein.

Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 238.43K | Year: 2008

DESCRIPTION (provided by applicant): A clinical need exists for better osteoporosis treatments since only one anabolic therapy (recombinant human parathyroid hormone) is commercially available. Bears are uniquely able to prevent bone loss during physical inactivity (hibernation) by maintaining balanced bone remodeling; the biological processes behind this phenomenon are likely governed by endogenous parathyroid hormone (PTH), since serum PTH levels are correlated with the bone formation marker osteocalcin in hibernating and active bears. Thus, our goal is to investigate the anabolic effects of bear PTH in comparison to human PTH to aid in the development of a more effective anabolic treatment for osteoporosis. It is possible to upregulate anabolic pathways in bone cells by changing PTH's amino acid sequence. Bear PTH has eleven differences in its amino acid sequence compared to human PTH. Thus, bear PTH may promote a greater anabolic response in bone than human PTH by decreasing osteoblast apoptosis and incr easing bone matrix production by altering gene expression. We propose to recombinantly produce bear PTH 1-84 and measure its ability to promote bone formation in vitro by measuring bear PTH's effects on apoptosis and gene expression in bone cells (MC-3T3 o steoblastic cells). The cellular responses will be compared with the effects of recombinant human PTH 1-84 to determine the relative anabolic potential of bear PTH. We will also treat rats with daily subcutaneous injections of human PTH 1-84 and bear PTH 1 -84. Femoral bone strength will be determined by 3-point bending tests and vertebral bone strength by compression tests. Bone formation rate will be quantified histologically in calcein labeled bone tissue, mineral content will be determined by ashing, and geometry (e.g., cross- sectional area) will be quantified by image analysis techniques. PUBLIC HEALTH RELEVENCE: Improved drug therapies are needed to treat the approximately 44 million Americans affected by osteoporosis. Bears uniquely prevent osteoporo sis during physical inactivity (hibernation) through biological processes regulated by parathyroid hormone (PTH). Injections of human PTH are currently used to rebuild bone in severely osteoporotic patients, but due to its different protein structure, bear PTH may cause greater bone formation than human PTH, and therefore may be a more effective treatment for human osteoporosis.

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