• 1,25-(OH)2-vitamin D3 suppresses the bone-related Runx2/Cbfa1 gene promoter

      Drissi, Hicham; Pouliot, Arlyssa; Koolloos, Christian; Stein, Janet L.; Lian, Jane B.; Stein, Gary S.; Van Wijnen, Andre J. (2002-03-20)
      The steroid hormone 1,25-(OH)2-vitamin D3 (VD3) regulates osteoblast differentiation by either activating or repressing transcription of numerous bone phenotypic genes. We addressed whether VD3 also influences osteogenesis by controlling activity of the runt-related transcription factor Runx2/Cbfa1, a key regulator of bone formation in vivo. Our data showed that expression of Runx2 was downregulated by VD3 within 24 h in MC3T3 and ROS 17/2.8, but not in ROS 24.1 cells, which lack a functional vitamin D receptor (VDR). Transient transfection assays showed that the initial 0.6 kb of the bone-related rat and mouse Runx2 promoters both exhibited a 50% reduction of promoter activity in response to VD3 in osteoblastic cells. Furthermore, VD3 inhibited Runx2 transcription in ROS 24.1 cells only upon forced expression of the VDR. Gel mobility shift assays with antibodies and oligonucleotide competition experiments demonstrated that proximal promoter sequences (-92 to -16) contain a functional VD3-responsive element (VDRE) that binds a VDR/retinoid X receptor heterodimer. Mutation of this VDRE completely abolished responsiveness of the Runx2 promoter to VD3 treatment. Together these studies establish that Runx2 expression is regulated by VD3. This VD3-mediated suppression of Runx2 activity provides regulatory coupling between tissue-specific and steroid hormone-dependent control of genes during bone formation.
    • 1801: Patient-Specific Markers Associated with the Risk of Alcohol Withdrawal in the Trauma Population

      Li, Irene; Forni, Allison; Carpenter, Dawn; Menard, Alexander; Rossetti, Victoria; Emhoff, Timothy A.; Lilly, Craig M. (2019-01-01)
      Learning Objectives: Alcohol withdrawal syndrome (AWS) and delirium tremens (DTs) have been reported as a complication following traumatic injury, at rates of 0.88% and 0.10%, respectively. AWS among trauma patients is associated with an increased length of hospital stay, mechanical ventilation, and aspiration pneumonia. There is minimal literature about the optimal screening tool to predict and stratify trauma patients at risk for AWS. Additionally, there has been increased interest in the use of phenobarbital as a management strategy for AWS. The purpose of this systematic review were to evaluate screening tools to identify trauma patients who are most likely to develop AWS, and assess phenobarbital dosing strategies for prophylaxis of AWS. Methods: A literature search was performed in PubMed/MEDLINE. The initial search yielded 1072 articles from which non-English and duplicate articles were removed. The remaining 974 articles underwent blinded review with an interprofessional team. Twenty articles were included in the final analysis. These studies were assessed for level of evidence using the Grading of Recommendations Assessment, Development and Evaluation tool (GRADE). Results: Twenty articles were included in the final analysis with eleven reviewing tools and nine reviewing phenobarbital dosing strategies. CAGE and brief MAST were the most commonly used questionnaires for adults with blunt or penetrating trauma. Other factors associated with the development of AWS and DTs include male gender, age, and blood alcohol level. Laboratory biomarkers such as elevated AST and MCV are associated with alcohol use among trauma victims. While phenobarbital has been evaluated for management of AWS and DTs, none of the studies utilized phenobarbital in the prevention of AWS or DTs. Conclusions: Further investigation into the predictive ability of a screening tool that combines a short questionnaire, laboratory values, and patient demographics to predict and stratify the risk of AWS and DTs in the adult trauma population is warranted. Additional research is needed to identify pharmacologic strategies for prophylaxis of AWS and DTs.
    • 1alpha,25-dihydroxy vitamin D3-enhanced expression of the osteocalcin gene involves increased promoter occupancy of basal transcription regulators and gradual recruitment of the 1alpha,25-dihydroxy vitamin D3 receptor-SRC-1 coactivator complex

      Carvallo, Loreto; Henriquez, Berta; Paredes, Roberto; Olate, Juan; Onate, Sergio; Van Wijnen, Andre J.; Lian, Jane B.; Stein, Gary S.; Stein, Janet L.; Montecino, Martin A. (2007-09-06)
      Binding of 1alpha,25-dihydroxy vitamin D(3) to the C-terminal ligand-binding domain (LBD) of its receptor (VDR) induces a conformational change that enables interaction of VDR with transcriptional coactivators such as members of the p160/SRC family or the DRIP (vitamin D receptor-interacting complex)/Mediator complex. These interactions are critical for VDR-mediated transcriptional enhancement of target genes. The p160/SRC members contain intrinsic histone acetyl transferase (HAT) activities that remodel chromatin at promoter regulatory regions, and the DRIP/Mediator complex may establish a molecular bridge between the VDR complex and the basal transcription machinery. Here, we have analyzed the rate of recruitment of these coactivators to the bone-specific osteocalcin (OC) gene in response to short and long exposures to 1alpha,25-dihydroxy vitamin D3. We report that in intact osteoblastic cells VDR, in association with SRC-1, rapidly binds to the OC promoter in response to the ligand. The recruitment of SRC-1 correlates with maximal transcriptional enhancement of the OC gene at 4 h and with increased histone acetylation at the OC promoter. In contrast to other 1alpha,25-dihydroxy vitamin D3-enhanced genes, binding of the DRIP205 subunit, which anchors the DRIP/Mediator complex to the VDR, is detected at the OC promoter only after several hours of incubation with 1alpha,25-dihydroxy vitamin D(3), concomitant with the release of SRC-1. Together, our results support a model where VDR preferentially recruits SRC-1 to enhance bone-specific OC gene transcription.
    • 20+ yr without leukemic or fibrotic transformation in essential thrombocythemia or polycythemia vera: predictors at diagnosis

      Tefferi, Ayalew; Gangat, Naseema; Wolanskyj, Alexandra P.; Schwager, Susan M.; Pardanani, Animesh Dev; Lasho, Terra L.; Mesa, Ruben A.; McClure, Rebecca F.; Li, Chin-Yang; Hanson, Curtis A. (2008-01-29)
      OBJECTIVES: The current study identified patients with either essential thrombocythemia (ET) or polycythemia vera (PV) who have survived for at least 20 yr without the development of either acute myeloid leukemia/myelodysplastic syndrome (AML/MDS) or myelofibrosis (MF) and compared their presenting features with those in whom these complications occurred in the first 10 yr of disease. METHODS: The study patients were selected from an institutional database of 1061 patients with either ET (n = 603) or PV (n = 458). In both instances, three distinct groups were delineated and their presenting features compared; group A included patients who have remained AML/MDS/MF free after a minimum follow-up of 20 yr; groups B and C included patients who developed either AML/MDS or MF, respectively, in the first decade of their disease. RESULTS: The respective number of patients who fulfilled the above-mentioned criteria for inclusion in groups A, B and C were 40, 12 and 8 for ET and 23, 18 and 12 for PV. In ET, compared with both groups B and C, group A displayed significantly fewer patients with less than normal hemoglobin level (P < 0.0001 and =0.02) or male sex (P = 0.005 and 0.05), respectively. On multivariable analysis, only anemia sustained its significance. A similar analysis in PV revealed an association between group B and leukocytosis using a leukocyte count threshold of either 10 or 15 x 10(9)/L (P = 0.02). CONCLUSION: The current study identifies PV patients with leukocytosis and ET patients with anemia as the most likely to undergo leukemic or fibrotic transformation.
    • 2019 Hope Babette Tang Humanism in Healthcare Essay Contest: First Place Nursing Student Essay

      Schultz, Kathleen (2019-12-01)
      The Arnold P. Gold Foundation holds an annual essay contest to encourage medical and nursing students to reflect on their experiences and engage in narrative writing. The contest began in 1999 focused on medical students and expanded to include nursing students in 2018. Students are asked to respond to a specific prompt in a 1,000-word essay. For the 2019 contest, students were asked to reflect on the following quote and share an experience in which they or their health care team engaged compassionately and respectfully with a patient to help them feel accepted and seen. "I long, as does every human being, to be at home wherever I find myself." —Maya Angelou. More than 300 essays were submitted. A distinguished panel of judges, ranging from esteemed medical professionals to notable authors, reviewed the submissions. Three winning essays from medical students and three winning essays from nursing students were selected, along with 10 honorable mentions. The winning essays will be published in consecutive issues of Academic Medicine and the Journal of Professional Nursing in the fall/winter of 2019. The contest is named for Hope Babette Tang-Goodwin, MD, who was an assistant professor of pediatrics. Her approach to medicine combined a boundless enthusiasm for her work, intellectual rigor, and deep compassion for her patients. She was an exemplar of humanism in medicine. The Arnold P. Gold Foundation infuses the human connection into health care. The nonprofit organization engages schools, health systems, companies, and individual clinicians in the joy and meaning of humanistic health care, so that they have the strength and knowledge to ensure that patients and families are partners in collaborative, compassionate, and scientifically excellent care.
    • 2020 Hindsight

      Wada-Gill, Bronwyn; Hansen, Megan (2021-01-21)
      Bronwyn Wada-Gill and Megan Hansen, University of Massachusetts Medical School (UMMS) students in the Class of 2022, solicited creative works to commemorate the year 2020 through looking back. The 2020 Hindsight website explores the words, music, and photography that came from the collective hearts of the UMMS community.
    • 3,4-Dichloropropionanilide (DCPA) inhibits T-cell activation by altering the intracellular calcium concentration following store depletion

      Lewis, Tricia L.; Brundage, Kathleen M.; Brundage, Rodney Arthur; Barnett, John B. (2008-02-19)
      Stimulation of T cells through the T-cell receptor results in the activation of a series of signaling pathways that leads to the secretion of interleukin (IL)-2 and cell proliferation. Influx of calcium (Ca(2+)) from the extracellular environment, following internal Ca(2+) store depletion, provides the elevated and sustained intracellular calcium concentration ([Ca(2+)](i)) critical for optimal T-cell activation. Our laboratory has documented that exposure to the herbicide 3,4-dichloropropionanilide (DCPA) inhibits intracellular signaling events that have one or more Ca(2+) dependent steps. Herein we report that DCPA attenuates the normal elevated and sustained [Ca(2+)](i) that follows internal store depletion in the human leukemic Jurkat T cell line and primary mouse T cells. DCPA did not alter the depletion of internal Ca(2+) stores when stimulated by anti-CD3 or thapsigargin demonstrating that early inositol 1,4,5-triphosphate-mediated signaling and depletion of Ca(2+) stores were unaffected. 2-Aminoethyldiphenol borate (2-APB) is known to alter the store-operated Ca(2+) (SOC) influx that follows Ca(2+) store depletion. Exposure of Jurkat cells to either DCPA or 50 microM 2-APB attenuated the increase in [Ca(2+)](i) following thapsigargin or anti-CD3 induced store depletion in a similar manner. At low concentrations, 2-APB enhances SOC influx but this enhancement is abrogated in the presence of DCPA. This alteration in [Ca(2+)](i), when exposed to DCPA, significantly reduces nuclear levels of nuclear factor of activated T cells (NFAT) and IL-2 secretion. The plasma membrane polarization profile is not altered by DCPA exposure. Taken together, these data indicate that DCPA inhibits T-cell activation by altering Ca(2+) homeostasis following store depletion.
    • 5' avian leukosis virus sequences and osteopetrotic potential

      Robinson, Harriet L.; Foster, Rosalinda Gram; Blais, Bruce P.; Reinsch, Sigrid S.; Newstein, Michael C.; Shank, Peter R. (1992-10-01)
      Recombinants of Rous-associated virus-0 and Br21 have been used to localize 5' viral sequences that affect the osteopetrotic potential of avian leukosis viruses. Rous-associated virus-0 is a benign subgroup E virus of endogenous origin that does not cause osteopetrosis. Br21 is a constructed subgroup E virus with high osteopetrotic potential. 5' sequences that affected osteopetrotic potential resided in an 834-bp region near the 5' LTR. Sequence analysis of this region revealed differences between Br21 and RAV-0 in the mRNA leader and codons for MA.
    • 6-Anilinouracil-based inhibitors of Bacillus subtilis DNA polymerase III: antipolymerase and antimicrobial structure-activity relationships based on substitution at uracil N3

      Tarantino, Paul M.; Zhi, Chengxin; Gambino, Joseph; Wright, George E.; Brown, Neal C. (1999-06-04)
      6-Anilinouracils (6-AUs) are dGTP analogues which selectively inhibit the DNA polymerase III of Bacillus subtilis and other Gram-positive bacteria. To enhance the potential of the 6-AUs as antimicrobial agents, a structure-activity relationship was developed involving substitutions of the uracil N3 position in two 6-AU platforms: 6-(3,4-trimethyleneanilino)uracil (TMAU) and 6-(3-ethyl-4-methylanilino)uracil (EMAU). Series of N3-alkyl derivatives of both 6-AUs were synthesized and tested for their ability to inhibit purified B. subtilis DNA polymerase III and the growth of B. subtilis in culture. Alkyl groups ranging in size from ethyl to hexyl enhanced the capacity of both platforms to bind to the polymerase, and with the exception of hexyl, they also significantly enhanced their antimicrobial potency. N3 substitution of the EMAU platform with more hydrophilic hydroxyalkyl and methoxyalkyl groups marginally enhanced anti-polymerase III activity but enhanced antibacterial potency severalfold. In sum, the results of these studies indicate that the ring N3 of 6-anilinouracils can tolerate substituents of considerable size and structural variety and, thus, can be manipulated to significantly enhance the antibacterial potency of this novel class of polymerase III-specific inhibitors.
    • A 'BOLD' experiment in defining the utility of fMRI in drug development

      Borsook, David; Bleakman, David; Hargreaves, Richard J.; Upadhyay, Jaymin; Schmidt, Karl F.; Becerra, Lino R. (2008-07-05)
    • A bayesian MCMC approach to assess the complete distribution of fitness effects of new mutations: uncovering the potential for adaptive walks in challenging environments

      Bank, Claudia; Hietpas, Ryan T.; Wong, Alex; Bolon, Daniel N.; Jensen, Jeffrey D. (2014-03-01)
      The role of adaptation in the evolutionary process has been contentious for decades. At the heart of the century-old debate between neutralists and selectionists lies the distribution of fitness effects (DFE)--that is, the selective effect of all mutations. Attempts to describe the DFE have been varied, occupying theoreticians and experimentalists alike. New high-throughput techniques stand to make important contributions to empirical efforts to characterize the DFE, but the usefulness of such approaches depends on the availability of robust statistical methods for their interpretation. We here present and discuss a Bayesian MCMC approach to estimate fitness from deep sequencing data and use it to assess the DFE for the same 560 point mutations in a coding region of Hsp90 in Saccharomyces cerevisiae across six different environmental conditions. Using these estimates, we compare the differences in the DFEs resulting from mutations covering one-, two-, and three-nucleotide steps from the wild type--showing that multiple-step mutations harbor more potential for adaptation in challenging environments, but also tend to be more deleterious in the standard environment. All observations are discussed in the light of expectations arising from Fisher's geometric model.
    • A Beta Test of a Computer-Assisted Instruction Module in Improving Dermatologic Physical Exam Skills in Third Year Medical Students

      Baldor, Robert; Domino, Frank; Deligiannidis, Konstantinos (2004-06-01)
      Introduction: Third year medical students at UMass. Medical School have only a brief exposure to dermatology during their first two years, resulting in limited skills in dermatology physical diagnosis. While other skills of the physical exam – e.g. the cardiac exam, the abdominal exam – are taught in the first 2 years and are applied and refined with repetition and instruction during students’ clinical rotations, dermatology remains limited in its exposure and instruction during the third year. This may prevent students from gaining the needed physical diagnosis skills in this important clinical area. Given this scenario, a computer-assisted instruction module was created and beta-tested to improve students’ physical diagnosis skills in dermatology. Method:A computer-based dermatology physical diagnosis presentation was created using Microsoft PowerPoint to instruct third year medical students in dermatology physical exam skills. Each slide presented students with a dermatology physical exam finding, a brief description, and a picture. In order to test the students’ knowledge of dermatology physical exam findings, a 23-item multiple-choice exam was created using XCom Exam Composer. 9 questions dealt with knowledge of verbal descriptions of lesions, while 14 questions dealt with the identification of lesions. The test was administered both before and after the presentation to 15 students taking a required 6-week Family Medicine clerkship at UMass. Medical School. Results:13 out of the 15 sampled students completed the curriculum. The average pre-test score was 18.4/23 questions correct (80%); the average post-test score was 20.5/23 questions correct (89.1%) - with an average improvement of 2.1 points (9.1%). These results (i.e., the increase in scores post-test) were statistically significant using both a parametric paired t-test (t = 2.720; p=.019) as well as a non-parametric Wilcoxon Signed Ranks Test to take the small sample size into account (z = 2.508; p=.012). Discussion: This computer-assisted instruction in dermatology significantly improved students’ physical exam skills, as measured by this multiple-choice exam. Informal comments were positive: students enjoyed the module, and appreciated the increased exposure to dermatology. Future studies are needed to evaluate student experience during this module, as well as their competence and confidence in diagnosing dermatologic lesions. Also, the Family Medicine Clerkship faculty intends to evaluate this module with a larger population of students, including those at different stages of their third year medical training, in order to examine the module’s effects. Furthermore, studies using a larger population are needed to evaluate the benefit and cost-effectiveness of this computer-assisted instruction program versus traditional book-based instruction.
    • A Biochemical Dissection of the RNA Interference Pathway in <em>Drosophila melanogaster</em>: A Dissertation

      Phillip D. Zamore, Ph. D.; Haley, Benjamin (2005-08-24)
      In diverse eukaryotic organisms, double-stranded RNA (dsRNA) induces robust silencing of cellular RNA cognate to either strand of the input dsRNA; a phenomenon now known as RNA interference (RNAi). Within the RNAi pathway, small, 21 nucleotide (nt) duplexed RNA, dubbed small interfering RNAs (siRNAs), derived from the longer input dsRNA, guide the RNA induced silencing complex (RISC) to destroy its target RNA. Due to its ability to silence virtually any gene, whether endogenous or exogenous, in a variety of model organisms and systems, RNAi has become a valuable laboratory tool, and is even being heralded as a potential therapy for an array of human diseases. In order to understand this complex and unique pathway, we have undertaken the biochemical characterization of RNAi in the model insect, Drosophila melanogaster. To begin, we investigated the role of ATP in the RNAi pathway. Our data reveal several ATP-dependent steps and suggest that the RNAi reaction comprises as least five sequential stages: ATP-dependent processing of double-stranded RNA into siRNAs, ATP-independent incorporation of siRNAs into an inactive ~360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, ATP-dependent activation of RISC following siRNA unwinding, and ATP-independent recognition and cleavage of the RNA target. In addition, ATP is used to maintain 5´ phosphates on siRNAs, and only siRNAs with these characteristic 5´ phosphates gain entry into the RNAi pathway. Next, we determined that RISC programmed exogenously with an siRNA, like that programmed endogenously with microRNAs (miRNAs), is an enzyme. However, while RISC behaves like a classical Michaelis-Menten enzyme in the presence of ATP, without ATP, multiple rounds of catalysis are limited by release of RISC-produced cleavage products. Kinetic analysis of RISC suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage and product release. Bases near the siRNA 5´ end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3´ region of the siRNA provide helical geometry required for catalysis. Lastly, the position of the scissile phosphate is determined during RISC assembly, before the siRNA encounters its RNA target. In the course of performing the kinetic assessment of RISC, we observed that when siRNAs are designed with regard to 'functional asymmetry' (by unpairing the 5´ terminal nucleotide of the siRNA's guide strand, i.e. the strand anti-sense to the target RNA), not all of the RISC formed was active for target cleavage. We observed, somewhat paradoxically, that increased siRNA unwinding and subsequent accumulation of single-stranded RNA into RISC led to reduced levels of active RISC formation. This inactive RISC did not act as a competitor for the active fraction. In order to characterize this non-cleaving complex, we performed a series of protein-siRNA photo-crosslinking assays. From these assays we found that thermodynamic stability and termini structure plays a role in determining which proteins an siRNA will associate with, and how association occurs. Furthermore, we have found, by means of the photo-crosslinking assays, that siRNAs commingle with components of the miRNA pathway, particularly Ago1, suggesting overlapping functions or crosstalk for factors thought to be involved in separate, distinct pathways.
    • A C. elegans genome-scale microRNA network contains composite feedback motifs with high flux capacity

      Martinez, Natalia Julia; Ow, Maria C.; Barrasa, M. Inmaculada; Hammell, Molly; Sequerra, Reynaldo; Doucette-Stamm, Lynn; Roth, Frederick P.; Ambros, Victor R.; Walhout, Albertha J. M. (2008-09-17)
      MicroRNAs (miRNAs) and transcription factors (TFs) are primary metazoan gene regulators. Whereas much attention has focused on finding the targets of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. Here, we present the first genome-scale Caenorhabditis elegans miRNA regulatory network that contains experimentally mapped transcriptional TF --> miRNA interactions, as well as computationally predicted post-transcriptional miRNA --> TF interactions. We find that this integrated miRNA network contains 23 miRNA <--> TF composite feedback loops in which a TF that controls a miRNA is itself regulated by that same miRNA. By rigorous network randomizations, we show that such loops occur more frequently than expected by chance and, hence, constitute a genuine network motif. Interestingly, miRNAs and TFs in such loops are heavily regulated and regulate many targets. This "high flux capacity" suggests that loops provide a mechanism of high information flow for the coordinate and adaptable control of miRNA and TF target regulons. Y1H dataset can be found as a supplemental file to this paper. See Additional Files below.
    • A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement

      Scanlon, Mary; Williams, David A.; Fay, Fredric S. (1987-05-05)
      The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.
    • A calcium-dependent protease as a potential therapeutic target for Wolfram syndrome

      Lu, Simin; Semenkovich, Clay F.; Greer, Peter A.; Urano, Fumihiko (National Academy of Sciences, 2014-12-09)
      Wolfram syndrome is a genetic disorder characterized by diabetes and neurodegeneration and considered as an endoplasmic reticulum (ER) disease. Despite the underlying importance of ER dysfunction in Wolfram syndrome and the identification of two causative genes, Wolfram syndrome 1 (WFS1) and Wolfram syndrome 2 (WFS2), a molecular mechanism linking the ER to death of neurons and β cells has not been elucidated. Here we implicate calpain 2 in the mechanism of cell death in Wolfram syndrome. Calpain 2 is negatively regulated by WFS2, and elevated activation of calpain 2 by WFS2-knockdown correlates with cell death. Calpain activation is also induced by high cytosolic calcium mediated by the loss of function of WFS1. Calpain hyperactivation is observed in the WFS1 knockout mouse as well as in neural progenitor cells derived from induced pluripotent stem (iPS) cells of Wolfram syndrome patients. A small-scale small-molecule screen targeting ER calcium homeostasis reveals that dantrolene can prevent cell death in neural progenitor cells derived from Wolfram syndrome iPS cells. Our results demonstrate that calpain and the pathway leading its activation provides potential therapeutic targets for Wolfram syndrome and other ER diseases.
    • A cell-based reglucosylation assay demonstrates the role of GT1 in the quality control of a maturing glycoprotein

      Pearse, Bradley R.; Gabriel, Luke; Wang, Ning; Hebert, Daniel N. (2008-04-23)
      The endoplasmic reticulum (ER) protein GT1 (UDP-glucose: glycoprotein glucosyltransferase) is the central enzyme that modifies N-linked carbohydrates based upon the properties of the polypeptide backbone of the maturing substrate. GT1 adds glucose residues to nonglucosylated proteins that fail the quality control test, supporting ER retention through persistent binding to the lectin chaperones calnexin and calreticulin. How GT1 functions in its native environment on a maturing substrate is poorly understood. We analyzed the reglucosylation of a maturing model glycoprotein, influenza hemagglutinin (HA), in the intact mammalian ER. GT1 reglucosylated N-linked glycans in the slow-folding stem domain of HA once the nascent chain was released from the ribosome. Maturation mutants that disrupted the oxidation or oligomerization of HA also supported region-specific reglucosylation by GT1. Therefore, GT1 acts as an ER quality control sensor by posttranslationally reglucosylating glycans on slow-folding or nonnative domains to recruit chaperones specifically to critical aberrant regions.
    • A central dinucleotide within vitamin D response elements modulates DNA binding and transactivation by the vitamin D receptor in cellular response to natural and synthetic ligands

      van den Bemd, Gert-Jan C. M.; Jhamai, Mila; Staal, Ada; Van Wijnen, Andre J.; Lian, Jane B.; Stein, Gary S.; Pols, Huibert A. P.; van Leeuwen, Johannes P. T. M. (2002-02-09)
      There is considerable divergence in the sequences of steroid receptor response elements, including the vitamin D response elements (VDREs). Two major VDRE-containing and thus 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3))-regulated genes are the two non-collagenous, osteoblast-derived bone matrix proteins osteocalcin and osteopontin. We observed a stronger induction of osteopontin than osteocalcin mRNA expression by 1,25-(OH)(2)D(3). Subsequently, we have shown that vitamin D receptor/retinoid X receptor alpha (VDR/RXRalpha) heterodimers bind more tightly to the osteopontin VDRE than to the osteocalcin VDRE. Studies using point mutants revealed that the internal dinucleotide at positions 3 and 4 of the proximal steroid half-element are most important for modulating the strength of receptor binding. In addition, studies with VDRE-driven luciferase reporter gene constructs revealed that the central dinucleotide influences the transactivation potential of VDR/RXRalpha with the same order of magnitude as that observed in the DNA binding studies. The synthetic vitamin D analog KH1060 is a more potent stimulator of transcription and inducer of VDRE binding of VDR/RXR in the presence of nuclear factors isolated from ROS 17/2.8 osteoblast-like cells than the natural ligand 1,25-(OH)(2)D(3). Interestingly, however, KH1060 is comparable or even less potent than 1,25-(OH)(2)D(3) in stimulating VDRE binding of in vitro synthesized VDR/RXRalpha. Thus, the extent of 1,25-(OH)(2)D(3)- and KH1060-dependent binding of VDR/RXRalpha is specified by a central dinucleotide in the VDRE, and the ligand-induced effects on DNA binding are in part controlled by the cellular context of nuclear proteins.
    • A Characterization of Substrates and Factors Involved in Yeast Nonsense-Mediated mRNA Decay: A Dissertation

      Dr. Allan Jacobson; Belk, Jonathan Philip (2002-01-08)
      Many intricate and highly conserved mechanisms have evolved to safeguard organisms against errors in gene expression. The nonsense-mediated mRNA decay pathway (NMD) exemplifies one such mechanism, specifically by eliminating mRNAs containing premature translation termination codons within their protein coding regions, thereby limiting the synthesis of potentially deleterious truncated polypeptides. Studies in Saccharomyces Cerevisiae have found that the activity of at least three trans-acting factors, known as UPF1, UPF2/NMD2, and UPF3is necessary for the proper function of the NMD pathway. Further research conducted in yeast indicates that the degradation of substrates of the NMD pathway is dependent on their translation, and that the sub-cellular site of their degradation in the cytoplasm. Although most evidence in yeast suggests that substrates of the NMD pathway are degraded in the cytoplasm while in association with the translation apparatus, some mammalian studies have found several mRNAs whose decay appears to occur within the nucleus or before their transport to the cytoplasm has been completed. In addition, study of the mammalian TPI mRNA found that this transcript was unavailable as a substrate for the NMD pathway once it had been successfully exported to the cytoplasm, further supporting the notion that the degradation of mammalian substrates of the NMD pathway occurs in association with the nucleus, or during export from the nucleus to the cytoplasm. To determine if yeast cytoplasmic nonsense-containing mRNA can become immune to the NMD pathway we examined the decay kinetics of two NMDS substrate mRNAs in response to repressing or activating the NMD pathway. Both the ade2-1 and pgk1-UAG-2nonsense-containing mRNAs were stabilized by repressing this pathway, while activation of NMD resulted in the rapid and immediate degradation of each transcripts. These findings demonstrate that nonsense-containing mRNAs residing in the nucleus are potentially susceptible to NMD at each round of translation. The remainder of this thesis utilizes protein overexpression studies to gain understanding into the function of factors related to the processes of nonsense-mediated mRNA decay and translation in Saccharomyces cerevisiae. Overexpression of a C-terminal truncated form of Nmd3p was found to be dominant-negative for cell viability, translation and the normal course of rRNA biogenesis. Overexpression studies conducted with mutant forms of the nonsense-mediated mRNA decay protein Upf1p, found that overexpression of mutants in the ATP binding and ATP hydrolysis region ofUpflp were dominant-negative for growth in an otherwise wild-type yeast strain. Furthermore, overexpression of the ATP hydrolysis mutant of Upf1p (DE572AA), resulted in the partial inhibition of NMD and a general perturbation of the translation apparatus. These results support previous studies suggesting a general role for Upf1p function in translation.
    • A chromatin landmark and transcription initiation at most promoters in human cells

      Guenther, Matthew G.; Levine, Stuart S.; Boyer, Laurie A.; Jaenisch, Rudolf; Young, Richard A. (2007-07-17)
      We describe the results of a genome-wide analysis of human cells that suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at postinitiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.