115 results on '"Wieben E"'
Search Results
2. Genetic diversity and function in the human cytosolic sulfotransferases
- Author
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Hildebrandt, M A T, Carrington, D P, Thomae, B A, Eckloff, B W, Schaid, D J, Yee, V C, Weinshilboum, R M, and Wieben, E D
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- 2007
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Catalog
3. Human catechol O-methyltransferase genetic variation: gene resequencing and functional characterization of variant allozymes
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Shield, A J, Thomae, B A, Eckloff, B W, Wieben, E D, and Weinshilboum, R M
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- 2004
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4. Human cytosolic sulfotransferase database mining: identification of seven novel genes and pseudogenes
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Freimuth, R R, Wiepert, M, Chute, C G, Wieben, E D, and Weinshilboum, R M
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- 2004
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5. Human sulfotransferase SULT2A1 pharmacogenetics: genotype-to-phenotype studies
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Thomae, B A, Eckloff, B W, Freimuth, R R, Wieben, E D, and Weinshilboum, R M
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- 2002
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6. OIII-A-3 HUMAN SULFOTRANSFERASE (SULT) 2A1 PHARMACOGENETICS: GENOTYPE TO PHENOTYPE STUDIES
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Thomae, B. A., Eckloff, B., Freimuth, R. R., Carlini, E., Wieben, E., and Weinshilboum, R. M.
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- 2001
7. PHARMACOGENETICS OF HUMAN SULFOTRANSFERASE (SULT) 1C1: GENE CLONING, RESEQUENCING AND COMMON SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs).
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Freimuth, R. R., Wood, T. C., Eckloff, B., Wieben, E., and Weinshilboum, R. M.
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- 2000
8. Gemcitabine pharmacogenomics: Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) gene sequence variation and functional genomics
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Eckloff, B. W., Ames, M. M., Kocabas, N. A., Yee, V. C., Gilbert, J. A., Salavaggione, O. E., Aksoy, P., Wieben, E. D., Weinshilboum, R. M., and Pelleymounter, L. L.
- Published
- 2008
9. Pharmacogenetics of the mycophenolic acid targets inosine monophosphate dehydrogenases IMPDH1 and IMPDH2: gene sequence variation and functional genomics.
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Wu, T-Y, Peng, Y, Pelleymounter, LL, Moon, I, Eckloff, BW, Wieben, ED, Yee, VC, Weinshilboum, RM, Pelleymounter, L L, Eckloff, B W, Wieben, E D, Yee, V C, and Weinshilboum, R M
- Subjects
PHARMACOGENOMICS ,MYCOPHENOLIC acid ,TARGETED drug delivery ,IMP dehydrogenase ,NUCLEOTIDE sequence ,HUMAN genetic variation ,FUNCTIONAL genomics ,RESEARCH ,BLACK people ,ANIMAL experimentation ,RESEARCH methodology ,GENETIC polymorphisms ,KIDNEY transplantation ,EVALUATION research ,MEDICAL cooperation ,ISOENZYMES ,NUCLEOTIDES ,PRIMATES ,COMPARATIVE studies ,RESEARCH funding ,IMMUNOSUPPRESSIVE agents ,OXIDOREDUCTASES ,WHITE people ,CELL lines ,PHARMACODYNAMICS ,CHEMICAL inhibitors - Abstract
Background and Purpose: Inosine monophosphate dehydrogenases, encoded by IMPDH1 and IMPDH2, are targets for the important immunosuppressive drug, mycophenolic acid (MPA). Variation in MPA response may result, in part, from genetic variation in IMPDH1 and IMPDH2.Experimental Approach: We resequenced IMPDH1 and IMPDH2 using DNA from 288 individuals from three ethnic groups and performed functional genomic studies of the sequence variants observed.Key Results: We identified 73 single nucleotide polymorphisms (SNPs) in IMPDH1, 59 novel, and 25 SNPs, 24 novel, in IMPDH2. One novel IMPDH1 allozyme (Leu275) had 10.2% of the wild-type activity as a result of accelerated protein degradation. Decreased activity of the previously reported IMPDH2 Phe263 allozyme was primarily due to decreased protein quantity, also with accelerated degradation. These observations with regard to the functional implications of variant allozymes were supported by the IMPDH1 and IMPDH2 X-ray crystal structures. A novel IMPDH2 intron 1 SNP, G > C IVS1(93), was associated with decreased mRNA quantity, possibly because of altered transcription.Conclusions and Implications: These results provide insight into the nature and extent of sequence variation in the IMPDH1 and IMPDH2 genes. They also describe the influence of gene sequence variation that alters the encoded amino acids on IMPDH function and provide a foundation for future translational studies designed to correlate sequence variation in these genes with outcomes in patients treated with MPA. [ABSTRACT FROM AUTHOR] more...- Published
- 2010
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10. Toxemia and fetal survival.
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McGUIRE, Kirk C., Keettel, William C., Wieben, Edward E., McGUIRE, K C, KEETTEL, W C, and WIEBEN, E E
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- 1954
11. Myosin individualized: single nucleotide polymorphisms in energy transduction
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Wieben Eric D, Neff Kevin L, Burghardt Thomas P, and Ajtai Katalin
- Subjects
Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Myosin performs ATP free energy transduction into mechanical work in the motor domain of the myosin heavy chain (MHC). Energy transduction is the definitive systemic feature of the myosin motor performed by coordinating in a time ordered sequence: ATP hydrolysis at the active site, actin affinity modulation at the actin binding site, and the lever-arm rotation of the power stroke. These functions are carried out by several conserved sub-domains within the motor domain. Single nucleotide polymorphisms (SNPs) affect the MHC sequence of many isoforms expressed in striated muscle, smooth muscle, and non-muscle tissue. The purpose of this work is to provide a rationale for using SNPs as a functional genomics tool to investigate structurefunction relationships in myosin. In particular, to discover SNP distribution over the conserved sub-domains and surmise what it implies about sub-domain stability and criticality in the energy transduction mechanism. Results An automated routine identifying human nonsynonymous SNP amino acid missense substitutions for any MHC gene mined the NCBI SNP data base. The routine tested 22 MHC genes coding muscle and non-muscle isoforms and identified 89 missense mutation positions in the motor domain with 10 already implicated in heart disease and another 8 lacking sequence homology with a skeletal MHC isoform for which a crystallographic model is available. The remaining 71 SNP substitutions were found to be distributed over MHC with 22 falling outside identified functional sub-domains and 49 in or very near to myosin sub-domains assigned specific crucial functions in energy transduction. The latter includes the active site, the actin binding site, the rigid lever-arm, and regions facilitating their communication. Most MHC isoforms contained SNPs somewhere in the motor domain. Conclusions Several functional-crucial sub-domains are infiltrated by a large number of SNP substitution sites suggesting these domains are engineered by evolution to be too-robust to be disturbed by otherwise intrusive sequence changes. Two functional sub-domains are SNP-free or relatively SNP-deficient but contain many disease implicated mutants. These sub-domains are apparently highly sensitive to any missense substitution suggesting they have failed to evolve a robust sequence paradigm for performing their function. more...
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- 2010
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12. 70% Renewables - Experiences and Solutions of a Large DSO in Northern Germany.
- Author
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Wieben, E.
- Subjects
DIGITAL storage oscilloscopes - Abstract
In this article, the author mentions about issues surrounding large digital storage oscilloscope in Northern Germany.
- Published
- 2014
13. 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer
- Author
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Poland Gregory A, Smith David I, Therneau Terry M, Oberg Ann L, Middha Sumit, Perez Edith A, Thompson E Aubrey, Klee Eric W, Asmann Yan W, Wieben Eric D, and Kocher Jean-Pierre A
- Subjects
Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract Background Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC). Results Using Brain RNA sample from multiple runs, we demonstrated that the transcript profiles from 3' DGE were highly reproducible between technical and biological replicates from libraries constructed by the same lab and even by different labs, and between two generations of Illumina's Genome Analyzers. Approximately 65% of all sequence reads mapped to mitochondrial genes, ribosomal RNAs, and canonical transcripts. The expression profiles of brain RNA and universal human reference RNA were compared which demonstrated that DGE was also highly quantitative with excellent correlation of differential expression with quantitative real-time PCR. Furthermore, one lane of 3' DGE sequencing, using the current sequencing chemistry and image processing software, had wider dynamic range for transcriptome profiling and was able to detect lower expressed genes which are normally below the detection threshold of microarrays. Conclusion 3' tag DGE profiling with massive parallel sequencing achieved high sensitivity and reproducibility for transcriptome profiling. Although it lacks the ability of detecting alternative splicing events compared to RNA-SEQ, it is much more affordable and clearly out-performed microarrays (Affymetrix) in detecting lower abundant transcripts. more...
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- 2009
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14. HUMAN METHYLENETETRAHYDROFOLATE REDUCTASE PHARMACOGENOMICS: GENE RESEQUENCING AND FUNCTIONAL GENOMIC STUDIES.
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Martin, Y. N., Salavaggione, O. E., Pelleymounter, L. L., Eckloff, B. W., Wieben, E. D., and Weinshilboum, R. M.
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- 2005
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15. Human phenylethanolamine N-methyltransferase (PNMT) pharmacogenetics: gene resequencing and functional genomic studies.
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Ji, Y., Salavaggione, O. E., Adjei, A. A., Thomae, B. A., Eckloff, B., Wieben, E. D., and Weinshilboum, R. M.
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- 2004
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16. Human Catecholamine Sulfotransferase (SULT1A3) Pharmacogenetics: Common Functional Genetic Polymorphism in African-American Subjects.
- Author
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Thomae, B., Rifki, O., Theobald, M., Eckloff, B., Wieben, E., and Weinshilboum, R.
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- 2003
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17. Sulfotransferase (sult) 1A1 pharmacogenetics: Functional 5′-flanking region (5′-FR) polymorphisms.
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Prondzinski, J., Thomae, B., Wang, L., Eckloff, B., Wieben, E., and Weinshilboum, R.
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- 2003
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18. The small nuclear ribonucleoprotein E protein gene contains four introns and has upstream similarities to genes for ribosomal proteins.
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Stanford, D R, Perry, C A, Holicky, E L, Rohleder, A M, and Wieben, E D
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- 1988
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19. Complete primary structure of a human plasma membrane Ca2+ pump.
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Verma, A K, Filoteo, A G, Stanford, D R, Wieben, E D, Penniston, J T, Strehler, E E, Fischer, R, Heim, R, Vogel, G, and Mathews, S
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- 1988
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20. Eukaryotic small ribonucleoproteins. Anti-La human autoantibodies react with U1 RNA-protein complexes.
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Madore, S J, Wieben, E D, and Pederson, T
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- 1984
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21. Expression of a secretory protein gene during androgen-induced cell growth.
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Moore, J T, Norvitch, M E, Wieben, E D, and Veneziale, C M
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- 1984
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22. MspI RFLP for SNRNPE gene on lq.
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Nishimura, D. Y., Wieben, E. D., Stanford, D. R., and Murray, J. C.
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- 1989
23. Conservation of coding and transcriptional control sequences within the snRNP E protein gene
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Wieben, E [Mayo Clinic/Foundation, Rochester, MN (United States)]
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- 1992
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24. Targeted long-read sequencing to quantify methylation of the C9orf72 repeat expansion.
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Udine E, Finch NA, DeJesus-Hernandez M, Jackson JL, Baker MC, Saravanaperumal SA, Wieben E, Ebbert MTW, Shah J, Petrucelli L, Rademakers R, Oskarsson B, and van Blitterswijk M
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- Humans, Male, Female, Middle Aged, Aged, Frontotemporal Dementia genetics, Sequence Analysis, DNA methods, C9orf72 Protein genetics, DNA Repeat Expansion genetics, DNA Methylation genetics, Amyotrophic Lateral Sclerosis genetics
- Abstract
Background: The gene C9orf72 harbors a non-coding hexanucleotide repeat expansion known to cause amyotrophic lateral sclerosis and frontotemporal dementia. While previous studies have estimated the length of this repeat expansion in multiple tissues, technological limitations have impeded researchers from exploring additional features, such as methylation levels., Methods: We aimed to characterize C9orf72 repeat expansions using a targeted, amplification-free long-read sequencing method. Our primary goal was to determine the presence and subsequent quantification of observed methylation in the C9orf72 repeat expansion. In addition, we measured the repeat length and purity of the expansion. To do this, we sequenced DNA extracted from blood for 27 individuals with an expanded C9orf72 repeat., Results: For these individuals, we obtained a total of 7,765 on-target reads, including 1,612 fully covering the expanded allele. Our in-depth analysis revealed that the expansion itself is methylated, with great variability in total methylation levels observed, as represented by the proportion of methylated CpGs (13 to 66%). Interestingly, we demonstrated that the expanded allele is more highly methylated than the wild-type allele (P-Value = 2.76E-05) and that increased methylation levels are observed in longer repeat expansions (P-Value = 1.18E-04). Furthermore, methylation levels correlate with age at collection (P-Value = 3.25E-04) as well as age at disease onset (P-Value = 0.020). Additionally, we detected repeat lengths up to 4,088 repeats (~ 25 kb) and found that the expansion contains few interruptions in the blood., Conclusions: Taken together, our study demonstrates robust ability to quantify methylation of the expanded C9orf72 repeat, capturing differences between individuals harboring this expansion and revealing clinical associations., Competing Interests: Declarations. Ethics approval and consent to participate: All subjects agreed to be in the study, and biological specimens were obtained after informed consent with approval from the Mayo Clinic Institutional Review Board (IRB). Consent for publication: Not applicable. Competing interests: MDJ and RR hold a patent on methods to screen for the C9orf72 hexanucleotide repeat expansion., (© 2024. The Author(s).) more...
- Published
- 2024
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25. Convulsive Status Epilepticus Induced by Electroconvulsive Therapy in a Patient with Major Depression.
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Wieben E, Kjeldsen MJ, and Sørensen CH
- Abstract
Electroconvulsive therapy (ECT) is a well-known, safe, and efficient treatment for a variety of psychiatric diseases. We present here an unusual case of a 34-year-old patient with major depression, who developed convulsive status epilepticus persistent for eight days in connection to her first ECT-a very uncommon but serious complication. The patient was, prior to ECT treatment, treated with lithium carbonate and clomipramine for her depression. Six years prior to the ECT, the patient had experienced a convulsive syncope resulting in traumatic subarachnoid haemorrhage. This case emphasizes the importance of medical recording to detect possible risk factors when considering ECT treatment., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Emilie Wieben et al.) more...
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- 2022
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26. Chromosomal Junction Detection from Whole-Genome Sequencing on Formalin-Fixed, Paraffin-Embedded Tumors.
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Murphy S, Smadbeck J, Eckloff B, Lee Y, Johnson S, Karagouga G, Serla V, Sharma A, Sikkink R, Voss J, Harris F, Kline JS, Kosari F, Feldman A, Wieben E, Aubry MC, Kipp B, Jen J, Cheville J, and Vasmatzis G more...
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- Algorithms, DNA Copy Number Variations, DNA, Neoplasm genetics, DNA, Neoplasm isolation & purification, Female, Genome, Human, Genomics methods, Humans, Male, Neoplasms pathology, Fixatives chemistry, Formaldehyde chemistry, Neoplasms genetics, Paraffin Embedding methods, Tissue Fixation methods, Translocation, Genetic genetics, Whole Genome Sequencing methods
- Abstract
DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (>500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (<2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected >90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.) more...
- Published
- 2021
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27. Familial chronic megacolon presenting in childhood or adulthood: Seeking the presumed gene association.
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Camilleri M, Wieben E, Eckert D, Carlson P, Hurley O'Dwyer R, Gibbons D, Acosta A, and Klee EW
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- Colon pathology, Colon physiopathology, Enteric Nervous System pathology, Female, Hirschsprung Disease pathology, Hirschsprung Disease physiopathology, Humans, Male, Megacolon pathology, Megacolon physiopathology, Pedigree, Exome Sequencing, Enteric Nervous System physiopathology, Hirschsprung Disease genetics, Megacolon genetics, Membrane Proteins genetics, Nerve Tissue Proteins genetics, Polymorphism, Single Nucleotide
- Abstract
Objective: We identified a pedigree over five generations with 49 members, some of whom had chronic megacolon presenting in adolescence or adulthood. We aimed to assess the genetic cause of chronic megacolon through clinical and DNA studies., Design: After ethical approval and informed consent, family members provided answers to standard bowel disease questionnaires, radiological or surgical records, and DNA (buccal mucosal scraping). Exome DNA sequencing of colon tissue or blood DNA from seven family members with colon or duodenal dilatation, or no megacolon (n = 1) was carried out. Sanger sequencing was performed in 22 additional family members to further evaluate candidate variants. The study focused on genes of potential relevance to enteric nerve (ENS) maturation and Hirschsprung's disease or megacolon, based on the literature (GFRA1, NKX2-1, KIF26A, TPM3, ACTG2, SCN10A, and C17orf107 [CHRNE]) and other genetic variants that co-segregated with megacolon in the six affected family members., Results: Information was available in all except five members alive at time of study; among 30 members who provided DNA, six had definite megacolon, one megaduodenum, seven significant constipation without bowel dilatation, and 16 normal bowel function by questionnaire. Among genes studied, SEMA3F (g.3:50225360A>G; c1873A>G) was found in 6/6 family members with megacolon. The SEMA3F gene variant was assessed as potentially pathogenic, based on M-CAP in silico prediction. SEMA3F function is associated with genes (KIT and PDGFRB) that impact intestinal pacemaker function., Conclusion: Familial chronic megacolon appears to be associated with SEMA3F, which is associated with genes impacting enteric nerve or pacemaker function., (© 2019 John Wiley & Sons Ltd.) more...
- Published
- 2019
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28. Myopathy With SQSTM1 and TIA1 Variants: Clinical and Pathological Features.
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Niu Z, Pontifex CS, Berini S, Hamilton LE, Naddaf E, Wieben E, Aleff RA, Martens K, Gruber A, Engel AG, Pfeffer G, and Milone M
- Abstract
Objective: The aim of this study is to identify the molecular defect of three unrelated individuals with late-onset predominant distal myopathy; to describe the spectrum of phenotype resulting from the contributing role of two variants in genes located on two different chromosomes; and to highlight the underappreciated complex forms of genetic myopathies., Patients and Methods: Clinical and laboratory data of three unrelated probands with predominantly distal weakness manifesting in the sixth-seventh decade of life, and available affected and unaffected family members were reviewed. Next-generation sequencing panel, whole exome sequencing, and targeted analyses of family members were performed to elucidate the genetic etiology of the myopathy., Results: Genetic analyses detected two contributing variants located on different chromosomes in three unrelated probands: a heterozygous pathogenic mutation in SQSTM1 (c.1175C>T, p.Pro392Leu) and a heterozygous variant in TIA1 (c.1070A>G, p.Asn357Ser). The affected fraternal twin of one proband also carries both variants, while the unaffected family members harbor one or none. Two unrelated probands (family 1, II.3, and family 3, II.1) have a distal myopathy with rimmed vacuoles that manifested with index extensor weakness; the other proband (family 2, I.1) has myofibrillar myopathy manifesting with hypercapnic respiratory insufficiency and distal weakness., Conclusion: The findings indicate that all the affected individuals have a myopathy associated with both variants in SQSTM1 and TIA1 , respectively, suggesting that the two variants determine the phenotype and likely functionally interact. We speculate that the TIA1 variant is a modifier of the SQSTM1 mutation. We identify the combination of SQSTM1 and TIA1 variants as a novel genetic defect associated with myofibrillar myopathy and suggest to consider sequencing both genes in the molecular investigation of myopathy with rimmed vacuoles and myofibrillar myopathy although additional studies are needed to investigate the digenic nature of the disease. more...
- Published
- 2018
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29. RYR1 causing distal myopathy.
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Laughlin RS, Niu Z, Wieben E, and Milone M
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- Adult, Creatine Kinase metabolism, DNA Mutational Analysis, Distal Myopathies diagnosis, Electromyography, Heterozygote, Humans, Jaw Abnormalities physiopathology, Male, Muscle, Skeletal pathology, Pedigree, Phenotype, Polymorphism, Single Nucleotide, Upper Extremity physiopathology, Exome Sequencing, Distal Myopathies genetics, Ryanodine Receptor Calcium Release Channel genetics
- Abstract
Background: Congenital myopathies due to ryanodine receptor (RYR1) mutations are increasingly identified and correlate with a wide range of phenotypes, most commonly that of malignant hyperthermia susceptibility and central cores on muscle biopsy with rare reports of distal muscle weakness, but in the setting of early onset global weakness., Methods: We report a case of a patient presenting with childhood onset hand stiffness and adult onset progressive hand weakness and jaw contractures discovered to have two variants in the RYR1 gene., Results: The patient manifested with distal upper limb weakness which progressed to involve the distal lower limb, proximal upper limb, as well as the face in addition to limited jaw opening. Creatine kinase was mildly elevated with EMG findings supporting a myopathy. Muscle biopsy showed features consistent with centronuclear myopathy. Whole exome sequencing revealed a novel heterozygous pathogenic variant in RYR1 (c.12315_12328delAGAAATCCAGTTCC, p.Glu4106Alafs*8), and a heterozygous missense variant (c.10648C>T, p.Arg3550Trp) of unknown significance in compound heterozygous state., Conclusion: We expand the spectrum of RYR1-related myopathy with the description of a novel phenotype in an adult patient presenting with hand weakness and suggest considering RYR1 analysis in the diagnosis of distal myopathies., (© 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.) more...
- Published
- 2017
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30. Common Oncogene Mutations and Novel SND1-BRAF Transcript Fusion in Lung Adenocarcinoma from Never Smokers.
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Jang JS, Lee A, Li J, Liyanage H, Yang Y, Guo L, Asmann YW, Li PW, Erickson-Johnson M, Sakai Y, Sun Z, Jeon HS, Hwang H, Bungum AO, Edell ES, Simon VA, Kopp KJ, Eckloff B, Oliveira AM, Wieben E, Aubry MC, Yi E, Wigle D, Diasio RB, Yang P, and Jen J more...
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- Adenocarcinoma metabolism, Adenocarcinoma pathology, Adenocarcinoma of Lung, Aged, Aged, 80 and over, Biomarkers, Tumor, Endonucleases, Extracellular Signal-Regulated MAP Kinases metabolism, Female, Gene Order, Humans, Lung Neoplasms metabolism, Lung Neoplasms pathology, Male, Middle Aged, Mitogen-Activated Protein Kinases metabolism, Neoplasm Staging, Nuclear Proteins metabolism, Oncogene Proteins, Fusion metabolism, Oncogenes, Phosphorylation, Proto-Oncogene Proteins B-raf metabolism, Reproducibility of Results, Transcription, Genetic, Adenocarcinoma genetics, Lung Neoplasms genetics, Mutation, Nuclear Proteins genetics, Oncogene Proteins, Fusion genetics, Proto-Oncogene Proteins B-raf genetics
- Abstract
Lung adenocarcinomas from never smokers account for approximately 15 to 20% of all lung cancers and these tumors often carry genetic alterations that are responsive to targeted therapy. Here we examined mutation status in 10 oncogenes among 89 lung adenocarcinomas from never smokers. We also screened for oncogene fusion transcripts in 20 of the 89 tumors by RNA-Seq. In total, 62 tumors had mutations in at least one of the 10 oncogenes, including EGFR (49 cases, 55%), K-ras (5 cases, 6%), BRAF (4 cases, 5%), PIK3CA (3 cases, 3%), and ERBB2 (4 cases, 5%). In addition to ALK fusions identified by IHC/FISH in four cases, two previously known fusions involving EZR- ROS1 and KIF5B-RET were identified by RNA-Seq as well as a third novel fusion transcript that was formed between exons 1-9 of SND1 and exons 2 to 3' end of BRAF. This in-frame fusion was observed in 3/89 tested tumors and 2/64 additional never smoker lung adenocarcinoma samples. Ectopic expression of SND1-BRAF in H1299 cells increased phosphorylation levels of MEK/ERK, cell proliferation, and spheroid formation compared to parental mock-transfected control. Jointly, our results suggest a potential role of the novel BRAF fusion in lung cancer development and therapy. more...
- Published
- 2015
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31. DPAGT1 myasthenia and myopathy: genetic, phenotypic, and expression studies.
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Selcen D, Shen XM, Brengman J, Li Y, Stans AA, Wieben E, and Engel AG
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- Adolescent, Child, Female, Humans, Intellectual Disability metabolism, Male, Muscle Weakness metabolism, Muscle, Skeletal metabolism, Mutation, Myasthenic Syndromes, Congenital metabolism, N-Acetylglucosaminyltransferases metabolism, Phenotype, Synapses genetics, Synapses metabolism, Intellectual Disability genetics, Muscle Weakness genetics, Myasthenic Syndromes, Congenital genetics, N-Acetylglucosaminyltransferases genetics
- Abstract
Objective: To investigate patients with DPAGT1 (UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase 1)-associated myasthenic syndrome., Methods: We performed exome and Sanger sequencing, determined glycoprotein expression in patient muscles, assessed pathogenicity of the mutant proteins by examining their expression and enzymatic activity in transfected cells, evaluated structural changes in muscle and the neuromuscular junction, and examined electrophysiologic aspects of neuromuscular transmission in vitro., Results: Patients 1 and 2, 16 and 14 years of age, had progressive fatigable weakness since infancy and are intellectually disabled. Patient 3, a less severely affected brother of patient 1, also has autistic features. Each patient harbors 2 novel heteroallelic mutations in DPAGT1, an enzyme subserving protein N-glycosylation. Patients 1 and 3 harbor Met1Leu, which reduces protein expression, and His375Tyr, which decreases enzyme activity. Patient 2 carries Val264Met, which abolishes enzyme activity, and a synonymous Leu120Leu mutation that markedly augments exon skipping, resulting in some skipped and infrequent nonskipped alleles. Therefore, the nonskipped allele rescues the phenotype. Intracellular microelectrode studies indicate combined pre- and postsynaptic defects of neuromuscular transmission with evidence for somatic mosaicism in patient 2. Structural studies reveal hypoplastic endplates, fiber-type disproportion, tubular aggregates, and degeneration of muscle fiber organelles resulting in autophagocytosis., Conclusions: DPAGT1 myasthenia affects multiple parameters of neuromuscular transmission, causes fiber-type disproportion and an autophagic myopathy, and can be associated with intellectual disability. We speculate that hypoglycosylation of synapse-specific proteins causes defects in central as well as motor synapses., (© 2014 American Academy of Neurology.) more...
- Published
- 2014
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32. Methionine adenosyltransferase 2A/2B and methylation: gene sequence variation and functional genomics.
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Nordgren KK, Peng Y, Pelleymounter LL, Moon I, Abo R, Feng Q, Eckloff B, Yee VC, Wieben E, and Weinshilboum RM
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- Animals, COS Cells, Chlorocebus aethiops, Exons, Humans, Methylation, Models, Molecular, Polymorphism, Single Nucleotide, Protein Interaction Domains and Motifs, Sequence Analysis, DNA methods, Methionine Adenosyltransferase genetics, Methionine Adenosyltransferase metabolism
- Abstract
Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine, the major biological methyl donor. MAT1A and MAT2A encode two distinct MAT isoforms in mammals. MAT2A is expressed in nonhepatic tissues, whereas MAT1A is expressed in the liver. A third gene, MAT2B, encodes a MAT2A regulatory protein. We resequenced MAT2A and MAT2B exons, splice junctions, and flanking regions using 288 DNA samples from three ethnic groups and also imputed additional single nucleotide polymorphisms (SNPs) across both genes using data from the 1000 Genomes Project. For MAT2A, resequencing identified 74 polymorphisms, including two nonsynonymous (ns) SNPs. Functional genomic studies of wild type and the two MAT2A variant allozymes (Val11 and Val205) showed that the Val11 allozyme had approximately 40% decreases in levels of enzyme activity and immunoreactive protein after COS-1 cell transfection. For MAT2B, 44 polymorphisms, 2 nonsynonymous, were identified during resequencing. Neither of the two MAT2B nsSNPs displayed alterations in levels of protein. Imputation using 1000 Genomes Project data resulted in 1730 additional MAT2A and 1997 MAT2B polymorphisms within ± 200 kilobases of each gene, respectively. Coexpression of MAT2A and MAT2B in COS-1 cells resulted in significantly increased MAT enzyme activity that correlated with increased MAT2A and MAT2B immunoreactive protein, apparently as a result of decreased degradation. Finally, studies of mRNA expression in lymphoblastoid cells showed that 7 SNPs in MAT2A and 16 SNPs in MAT2B were significantly associated with mRNA expression with p < 0.01. These observations provide a foundation for future mechanistic and clinical translational pharmacogenomic studies of MAT2A/2B. more...
- Published
- 2011
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33. SLC6A4 variation and citalopram response.
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Mrazek DA, Rush AJ, Biernacka JM, O'Kane DJ, Cunningham JM, Wieben ED, Schaid DJ, Drews MS, Courson VL, Snyder KA, Black JL 3rd, and Weinshilboum RM
- Subjects
- Adult, Black or African American genetics, Alleles, Clinical Trials as Topic, Depressive Disorder, Major drug therapy, Female, Gene Frequency, Genetic Variation, Haplotypes, Hispanic or Latino genetics, Humans, Introns, Linkage Disequilibrium, Male, Middle Aged, Minisatellite Repeats, Polymorphism, Single Nucleotide, Promoter Regions, Genetic, Remission Induction, Sequence Analysis, DNA, Treatment Outcome, White People genetics, Antidepressive Agents, Second-Generation therapeutic use, Citalopram therapeutic use, Depressive Disorder, Major genetics, Serotonin Plasma Membrane Transport Proteins genetics, Selective Serotonin Reuptake Inhibitors therapeutic use
- Abstract
The influence of genetic variations in SLC6A4 (serotonin transporter gene) on citalopram treatment of depression using the Sequenced Treatment to Relieve Depression (STAR*D) sample was assessed. Of primary interest were three previously studied polymorphisms: 1) the VNTR variation of the second intron, 2) the indel promoter polymorphism (5HTTLPR or SERT), and 3) a single nucleotide polymorphism (SNP) rs25531. Additionally, SLC6A4 was resequenced to identify new SNPs for exploratory analyses. DNA from 1914 subjects in the STAR*D study were genotyped for the intron 2 VNTR region, the indel promoter polymorphism, and rs25531. Associations of these variants with remission of depressive symptoms were evaluated following citalopram treatment. In white non-Hispanic subjects, variations in the intron 2 VNTR (point-wise P = 0.041) and the indel promoter polymorphism (point-wise P = 0.039) were associated with remission following treatment with citalopram. The haplotype composed of the three candidate loci was also associated with remission, with a global p-value of 0.040 and a maximum statistic simulation p-value of 0.0031 for the S-a-12 haplotype, under a dominant model. One SNP identified through re-sequencing the SLC6A4 gene, Intron7-83-TC, showed point-wise evidence of association, which did not remain significant after correction for the number of SNPs evaluated in this exploratory analysis. No associations between these SLC6A4 variations and remission were found in the white Hispanic or black subjects. These findings suggest that multiple variations in the SLC6A4 gene are associated with remission in white non-Hispanic depressed adults treated with citalopram. The mechanism of action of these variants remains to be determined., ((c) 2008 Wiley-Liss, Inc.) more...
- Published
- 2009
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34. Human 3beta-hydroxysteroid dehydrogenase types 1 and 2: Gene sequence variation and functional genomics.
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Wang L, Salavaggione E, Pelleymounter L, Eckloff B, Wieben E, and Weinshilboum R
- Subjects
- 3-Hydroxysteroid Dehydrogenases metabolism, 5' Flanking Region, Animals, COS Cells, Chlorocebus aethiops, Genes, Reporter, Genomics, Haplotypes, Humans, Linkage Disequilibrium, Microscopy, Confocal, Polymorphism, Single Nucleotide, Protein Structure, Tertiary, Reverse Transcriptase Polymerase Chain Reaction, 3-Hydroxysteroid Dehydrogenases genetics, Genetic Variation
- Abstract
The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase isoenzymes 1 and 2 (HSD3B1 and HSD3B2) are membrane-bound enzymes that play essential roles in the biosynthesis of steroid hormones. Therefore, variation in the HSD3B1 and HSD3B2 genes might play a role in the pathophysiology of steroid hormone-related disease. We set out to systematically identify common polymorphisms and haplotypes in human HSD3B1 and HSD3B2. We identified 17 single nucleotide polymorphisms (SNPs) in HSD3B1 and 9 in HSD3B2 - the majority of which were not present in public databases - by resequencing human HSD3B1 and HSD3B2 using 240 DNA samples from four different ethnic groups (60 samples per group). Functional genomic studies of the five non-synonymous cSNPs in HSD3B1 and the one observed in HSD3B2 showed that two of these polymorphisms resulted in significant decreases in the quantity of enzyme protein expressed. However, none of the three non-synonymous SNPs located in areas encoding putative membrane-binding domains altered subcellular localization of the enzyme as determined by immunofluorescence microscopy. Finally, common variant haplotypes in the 5'-flanking regions of these genes showed significant cell line-dependent variation in their ability to drive transcription. In aggregate, these results provide a basis for study of the possible role in human disease of common genetic variation in HSD3B1 and HSD3B2. more...
- Published
- 2007
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35. Genetic nondiscrimination legislation: a critical prerequisite for pharmacogenomics data sharing.
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Altman RB, Benowitz N, Gurwitz D, Lunshof J, Relling M, Lamba J, Wieben E, Mooney S, Giacomini K, Weiss S, Johnson JA, McLeod H, Flockhart D, Weinshilboum R, Shuldiner AR, Roden D, Krauss RM, and Ratain M more...
- Subjects
- Employment legislation & jurisprudence, Insurance legislation & jurisprudence, United States, Genetic Privacy legislation & jurisprudence, Government Regulation, Medical Records legislation & jurisprudence, Pharmacogenetics legislation & jurisprudence, Prejudice
- Published
- 2007
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36. Human histamine N-methyltransferase pharmacogenetics: gene resequencing, promoter characterization, and functional studies of a common 5'-flanking region single nucleotide polymorphism (SNP).
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Wang L, Thomae B, Eckloff B, Wieben E, and Weinshilboum R
- Subjects
- 5' Flanking Region genetics, DNA analysis, Genotype, Histamine metabolism, Histamine N-Methyltransferase physiology, Humans, Phenotype, Histamine N-Methyltransferase genetics, Polymorphism, Single Nucleotide, Promoter Regions, Genetic genetics
- Abstract
Histamine N-methyltransferase (HNMT) catalyzes one of two major metabolic pathways for histamine. The levels of HNMT activity and immunoreactive protein in human tissues are regulated primarily by inheritance. Previous studies of HNMT identified two common single nucleotide polymorphisms (SNPs), including a functionally significant nonsynonymous coding SNP (cSNP), (C314T, Thr105Ile), but that polymorphism did not explain all of the phenotypic variation. In the present study, a genotype-to-phenotype strategy was used to search for additional genetic factors that might contribute to the regulation of human HNMT activity. Specifically, we began by resequencing the human HNMT gene using 90 ethnically anonymous DNA samples from the Coriell Cell Repository and identified a total of eight SNPs, including the two that had been reported previously. No new nonsynonymous cSNPs were observed, but three of the six novel SNPs were located in the 5'-flanking region (5'-FR) of the gene-including a third common polymorphism with a frequency of 0.367 (36.7%). That observation directed our attention to possible genetic effects on HNMT transcription. As a first step in testing that possibility, we created and studied a series of reporter gene constructs for the initial 1kb of the HNMT 5'-FR. The core promoter and possible regulatory regions were identified and verified by electrophoresis mobility shift assays. We then studied the possible functional implications of the new common HNMT 5'-FR SNP. However, on the basis of reporter gene studies, that SNP appeared to have little effect on transcription. Phenotype-genotype correlation analysis performed with 112 human kidney biopsy samples that had been phenotyped for their level of HNMT activity confirmed that the common 5'-FR SNP was not associated with the level of HNMT activity in vivo. In summary, this series of experiments resulted in the identification of several novel HNMT polymorphisms, identification of the HNMT core promoter, and a comprehensive functional genomic study of a common HNMT 5'-FR SNP. These results represent an additional step in the definition of molecular genetic mechanisms involved in the regulation of this important autacoid-metabolizing enzyme in humans. more...
- Published
- 2002
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37. Human 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) pharmacogenetics: gene resequencing, genetic polymorphisms and functional characterization of variant allozymes.
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Xu ZH, Freimuth RR, Eckloff B, Wieben E, and Weinshilboum RM
- Subjects
- Animals, Base Sequence, Blotting, Western, COS Cells, DNA Primers chemistry, Humans, Isoenzymes genetics, Linkage Disequilibrium, Molecular Sequence Data, Pharmacogenetics, Polymerase Chain Reaction, Sequence Analysis, DNA, Transfection, Multienzyme Complexes genetics, Polymorphism, Single Nucleotide, Sulfate Adenylyltransferase genetics
- Abstract
3'-Phosphoadenosine 5'-phosphosulfate (PAPS) is the sulfate donor cosubstrate for all sulfotransferase (SULT) enzymes. SULTs catalyze the sulfate conjugation of many endogenous and exogenous compounds, including drugs and other xenobiotics. In humans, PAPS is synthesized from adenosine 5'-triphosphate (ATP) and inorganic sulfate (SO2-4) by two isoforms, PAPSS1 and PAPSS2. Rare mutations that inactivate PAPSS2 are associated with human spondyloepimetaphyseal dysplasia and murine brachymorphism. To determine whether more common genetic polymorphisms that do not completely inactivate the enzyme might be one factor responsible for individual differences in sulfate conjugation, we previously cloned the human PAPSS2 gene. In the present studies, we 'resequenced' all twelve PAPSS2 exons and splice junctions, as well as approximately 500 bp of the 5'-flanking region, using 90 Polymorphism Discovery Resource (PDR) DNA samples from the Coriell Cell Repository. Twenty-two single nucleotide polymorphisms (SNPs) were observed, including four nonsynonymous coding region SNPs (cSNPs) that altered the following amino acids: Glu10Lys, Met281Leu,Val291Met and Arg432Lys. We also observed four insertions/deletions, including one sample that was homozygous for an 81-bp deletion in the 5'-flanking region 286 bp upstream from the site of transcription initiation. Transient expression studies showed that two of the nonsynonymous cSNPS, those that resulted in Glu10Lys and Val291Met alterations in encoded amino acids, showed significant decreases in levels of PAPSS activity. In the case of Glu10Lys, decreased activity was paralleled by a decrease in immunoreactive protein, while the Val291Met allozyme displayed a significant decrease in affinity for both ATP and Na2SO4 when compared to 'wild-type' enzyme, but without a significant alteration in level of immunoreactive protein. It will now be possible to test the hypothesis that these common, functionally significant PAPSS2 genetic polymorphisms might contribute to variations in sulfate conjugation in vivo. more...
- Published
- 2002
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38. Human sulfotransferase SULT1C1 pharmacogenetics: gene resequencing and functional genomic studies.
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Freimuth RR, Eckloff B, Wieben ED, and Weinshilboum RM
- Subjects
- Animals, Base Sequence, Blotting, Western, COS Cells, Exons, Female, Gene Frequency, Genomics, Haplotypes, Humans, Introns, Kinetics, Linkage Disequilibrium, Male, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymorphism, Single Nucleotide, Sequence Analysis, DNA, Sulfotransferases metabolism, Transfection, beta-Galactosidase metabolism, Pharmacogenetics methods, Polymorphism, Genetic, Sulfotransferases genetics
- Abstract
Sulfotransferase (SULT) enzymes catalyze an important phase II reaction in the biotransformation of many drugs and other xenobiotics. We previously cloned the human SULT1C1 cDNA and gene as steps toward pharmacogenetic studies. We have now 'resequenced' the exons, portions of introns flanking exons and approximately 315 bp of the 5' flanking region of SULT1C1 in 89 DNA samples from Caucasian subjects to identify common genetic polymorphisms. Nineteen separate polymorphisms were observed, including four nonsynonymous coding region single nucleotide polymorphisms (cSNPs) and five insertions/deletions. These data were also used to determine and/or infer common SULT1C1 haplotypes. Three of the four nonsynonymous cSNPs had allele frequencies greater than 1%, including one with a frequency of 6.7%. Expression constructs were created for all of the nonsynonymous cSNPs observed, and those constructs were used to transfect COS-1 cells. Three of the four SULT1C1 variant allozymes had significantly reduced enzyme activity when compared with the wild-type enzyme. Among the variant allozymes, apparent Km values for 3'-phosphoadenosine 5'-phosphosulfate (PAPS), the sulfate donor for the reaction, varied 7-fold, and quantitative Western blot analysis showed variable levels of immunoreactive protein when compared to the wild-type enzyme. Therefore, mechanisms responsible for decreased activity involved both alterations in levels of enzyme protein and alterations in substrate kinetics. In summary, application of a 'genotype to phenotype' strategy has resulted in the identification of a series of functionally significant common genetic polymorphisms for SULT1C1. It will now be possible to evaluate the possible contribution of these polymorphisms to variation in the sulfate conjugation of drugs, other xenobiotics and/or disease pathophysiology. more...
- Published
- 2001
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39. Cyclin-dependent kinase 5 is expressed in both Sertoli cells and metaphase spermatocytes.
- Author
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Session DR, Fautsch MP, Avula R, Jones WR, Nehra A, and Wieben ED
- Subjects
- Animals, Cell Cycle, Cyclin-Dependent Kinase 5, Humans, Immunohistochemistry, Male, Mice, Sertoli Cells cytology, Spermatocytes cytology, Testis cytology, Tubulin analysis, Cyclin-Dependent Kinases analysis, Sertoli Cells enzymology, Spermatocytes enzymology, Testis enzymology
- Abstract
Objective: To gain insight into the function of cyclin-dependent kinase 5 (Cdk5) in spermatogenesis., Design: The expression of the Cdk5 protein was determined with the use of immunohistochemical and immunoblot analysis., Setting: Academic research laboratory., Animal(s): Adult mouse and archival human testicular tissue were used for the immunohistochemical analysis. Adult mice were used as the source of tissues for the immunoblot analysis., Intervention(s): The immunohistochemical analysis was performed with an anti-Cdk5 antibody. The double immunohistochemical analysis was performed with anti-Cdk5 and alpha-tubulin antibodies. Immunoblotting was used to examine multiple mouse tissues for Cdk5 expression., Main Outcome Measure(s): Analysis of Cdk5 protein distribution., Result(s): Cdk5 was localized specifically within the cytoplasm of Sertoli cells and meiotic metaphase germ cells. The double immunohistochemistry analysis demonstrated the co-localization of Cdk5 and alpha-tubulin within the Sertoli cells. Western blot analysis revealed a high level of expression of Cdk5 in the testicular lysate., Conclusion(s): The cyclin-dependent kinases are known regulators of the cell cycle; however, Cdk5 expression previously has been described in terminally differentiated cells of the brain. The present evidence of an association between Cdk5 and microfilaments of Sertoli cells and meiotic metaphase germ cells suggests a role of Cdk5 in both seminiferous tubule function and meiosis. more...
- Published
- 2001
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40. Novel messenger RNA and alternative promoter for murine acetylcholinesterase.
- Author
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Atanasova E, Chiappa S, Wieben E, and Brimijoin S
- Subjects
- Alternative Splicing genetics, Animals, Base Sequence, Mice, Molecular Sequence Data, Sequence Analysis, DNA, Acetylcholinesterase genetics, Promoter Regions, Genetic genetics, RNA, Messenger genetics
- Abstract
A portion of the 5'-flanking region of murine acetylcholinesterase was cloned from genomic DNA by 5'-rapid amplification of genomic ends, identified in a mouse genomic library, and sequenced. Multiple potential binding sites for universal and tissue-specific transcription factors were suggestive of a promoter region within this DNA sequence. Potential promoter activity was confirmed by coupling the new sequence to the open reading frame of a luciferase reporter gene in transient expression experiments with nerve and muscle cells. 5'-Rapid amplification of cDNA ends with templates from multiple sources revealed a novel transcription start site (at position -626, relative to translation start), located 32 bases downstream from a TATAA sequence. This start site appeared to mark a novel exon (1a) comprising 291 base pairs between positions -335 and -626, relative to the translation start. Supporting this conclusion, polymerase chain reactions with cDNA from mouse brain, heart, and other tissues, consistently amplified a transcript containing the exon 1a sequence fused to the invariant sequence beginning at position -22 in exon 2, but lacking exon 1. Northern blot analyses confirmed the in vivo expression of exon 1a-containing transcripts, especially in heart, brain, liver, and kidney. These results indicate that the murine acetylcholinesterase gene has a functioning alternative promoter that may influence expression of acetylcholinesterase in certain tissues. more...
- Published
- 1999
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41. Characterization of the mouse TGFbeta-inducible early gene (TIEG): conservation of exon and transcriptional regulatory sequences with evidence of additional transcripts.
- Author
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Fautsch MP, Vrabel A, Rickard D, Subramaniam M, Spelsberg TC, and Wieben ED
- Subjects
- 5' Untranslated Regions, Amino Acid Sequence, Animals, Base Sequence, Conserved Sequence, DNA, Complementary genetics, Early Growth Response Transcription Factors, Exons, Genes, Regulator, Humans, Kruppel-Like Transcription Factors, Mice, Molecular Sequence Data, Promoter Regions, Genetic, Sequence Homology, Nucleic Acid, Species Specificity, Transcription, Genetic, DNA-Binding Proteins genetics, Transcription Factors genetics
- Published
- 1998
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42. In vivo expression of a variant human U6 RNA from a unique, internal promoter.
- Author
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Tichelaar JW, Wieben ED, Reddy R, Vrabel A, and Camacho P
- Subjects
- Animals, Base Sequence, Cell Nucleus genetics, Chemical Precipitation, Gene Expression Regulation, Genetic Variation, HeLa Cells, Humans, Molecular Sequence Data, Oocytes metabolism, Organophosphates metabolism, RNA Caps chemistry, RNA Caps metabolism, RNA, Small Nuclear genetics, RNA, Small Nuclear metabolism, Ribonucleoproteins genetics, Ribonucleoproteins metabolism, Transcription, Genetic, Xenopus laevis, Promoter Regions, Genetic, RNA, Small Nuclear biosynthesis
- Abstract
We previously isolated a variant of the human U6 small nuclear RNA gene (87U6) and demonstrated that transcription of this gene is controlled by a novel internal promoter. It has now been shown that two blocks of sequence within the coding region are both necessary and sufficient to direct expression of 87U6 in transcription assays performed in vitro. In addition, 87U6 is expressed in vivo and can assemble into snRNP complexes. Specific primer extension assays on total RNA from HeLa cells shows that 87U6 RNA is present in these cells. Also, microinjection of plasmid encoded 87U6 genes into Xenopus laevis oocyte nuclei results in the expression of this variant RNA. Immunoprecipitation with anti-Sm antibodies suggests that 87U6 RNA assembles into a snRNP particle with U4 snRNA. Finally, the variant snRNA is capped with the U6 specific gamma-monomethyl phosphate cap when incubated in HeLa extracts. These data suggest that 87U6 RNA may function in the splicing process, in a manner similar to the wild-type U6 RNA. The recent observations of a minor class of mRNA introns that are spliced by a distinct collection of snRNP particles suggest an important role for variant snRNAs in the splicing of transcripts with alternative splice junctions. more...
- Published
- 1998
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43. TGFbeta-inducible early gene (TIEG) also codes for early growth response alpha (EGRalpha): evidence of multiple transcripts from alternate promoters.
- Author
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Fautsch MP, Vrabel A, Subramaniam M, Hefferen TE, Spelsberg TC, and Wieben ED
- Subjects
- Base Sequence, Cloning, Molecular, Early Growth Response Transcription Factors, Exons genetics, Fetus metabolism, Genes, Reporter, Growth Substances pharmacology, Humans, Kruppel-Like Transcription Factors, Molecular Sequence Data, RNA Splicing genetics, RNA, Messenger metabolism, Sequence Analysis, DNA, Transfection, Zinc Fingers genetics, DNA-Binding Proteins genetics, Osteoblasts metabolism, Promoter Regions, Genetic genetics, Transcription Factors genetics, Transcription, Genetic genetics
- Abstract
TGFbeta-inducible early gene (TIEG) and early growth response alpha (EGRalpha) are putative transcription factors based on homology to known zinc finger proteins SP1, EGR1, BTEB, and Wilm tumor. Here we report that TIEG and EGRalpha are expressed from alternative promoters of the same gene. The TIEG/EGRalpha gene spans 8 kb and contains five exons. Use of alternative first exons results in TIEG having 12 unique amino acids on its N-terminus. Computer analysis of the 5' upstream regions of either TIEG (exon 1a) or EGRalpha (exon 1b) does not identify a TATA box or initiator sequencebut shows consensus sequence similarities to binding sites for several transcription factors including SP1,JunB, and aromatic hydrocarbon/receptor-ligand complexes. Analysis of constructs containing 5'-flanking regions show that both the TIEG and the EGRalpha promoters have significant activity in human fetal osteoblast cells. Northern analysis of mRNA from various human tissues and several cell lines reveals that TIEG is the predominant transcript produced and regulated by growth factors from the TIEG/EGRalpha gene., (Copyright 1998 Academic Press.) more...
- Published
- 1998
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44. Production of SVP-1/-3/-4 in guinea pig testis. Characterization of novel transcripts containing long 5'-untranslated regions and multiple upstream AUG codons.
- Author
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Fautsch MP, Perdok MM, and Wieben ED
- Subjects
- Animals, Base Sequence, DNA, Complementary, Gene Expression Regulation, Guinea Pigs, Male, Molecular Sequence Data, Proteins genetics, Sequence Homology, Nucleic Acid, Codon, Protein Biosynthesis, RNA, Messenger genetics, Seminal Vesicle Secretory Proteins, Testis metabolism
- Abstract
The GP1G gene of the guinea pig codes for three of the four abundant seminal vesicle secretory proteins produced in this species. This gene is expressed at highest efficiency in the seminal vesicle (SV) from a promoter that contains a canonical TATA box and CCAAT box. However, GP1G gene transcripts and proteins have also been identified in other tissues. To investigate the structure of GP1G transcripts produced in the testis, cDNA clones were isolated by screening a testis library. Three unique cDNAs (TSM1-3) were isolated. Each of these clones contained a 3'-untranslated region (UTR) and coding region identical to that of the seminal vesicle transcript. However, the 5'-UTRs of the testis transcripts were significantly longer than that found on the SV mRNA (416-646 nucleotides compared with only 23 nucleotides for the SV). Each of these alternatively spliced 5'-UTRs incorporated the SV promoter elements into transcribed sequence, and each contained multiple upstream AUG codons predicted to abolish translation of the major open reading frame. Nevertheless, each of the testis transcripts was capable of directing the synthesis of GP1G-related proteins in vitro. Analysis of the translation products suggests that the extended 5'-UTR of the testis transcripts regulate both the choice of translation start site and the efficiency of translation in this system. Western blot analysis of testis proteins revealed that the protein products of GP1G are also synthesized by the testis in vivo. more...
- Published
- 1997
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45. Structure of the rat collagen IV promoter.
- Author
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Grande JP, Melder DC, Kluge DL, and Wieben ED
- Subjects
- Animals, Base Sequence, Chloramphenicol O-Acetyltransferase genetics, Cloning, Molecular, Exons genetics, Genes genetics, Genes, Reporter genetics, Glomerular Mesangium cytology, Molecular Sequence Data, Rats, Recombinant Fusion Proteins, Transcription, Genetic genetics, Transfection, Collagen genetics, Promoter Regions, Genetic genetics
- Abstract
We have isolated a 1.6 kb clone from a rat genomic library which contains the bidirectional collagen IV promoter, flanked by exons coding for the alpha 1 (IV) and alpha 2 (IV) collagen chains. There are at least two transcription start sites within both the alpha 1 (IV) and alpha 2 (IV) collagen genes. Rat mesangial cells were transfected with chloramphenicol acetyltransferase (CAT) reporter plasmids containing segments of the promoter and 5' flanking region, in both the alpha 1 (IV) and alpha 2 (IV) orientations. Our results suggest that transcriptional efficiency of the bidirectional promoter is more efficient in the alpha 2 (IV) direction than in the alpha 1 (IV) direction. more...
- Published
- 1996
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46. Exons lost and found. Unusual evolution of a seminal vesicle transglutaminase substrate.
- Author
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Hagstrom JE, Fautsch MP, Perdok M, Vrabel A, and Wieben ED
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, DNA, Guinea Pigs, Humans, Male, Molecular Sequence Data, Sequence Homology, Amino Acid, Substrate Specificity, Biological Evolution, Exons, Proteins genetics, Proteins metabolism, Seminal Vesicle Secretory Proteins, Seminal Vesicles enzymology, Transglutaminases metabolism
- Abstract
The GP1G gene codes for three of the four abundant androgen-regulated secretory proteins produced by the guinea pig seminal vesicle. Sequencing of the entire 6.3-kilobase gene and comparison with other mammalian seminal vesicle secretory protein genes reveals a common three-exon, two-intron organization. However, significant sequence similarity between this group of genes is largely limited to their 5'-flanking regions and first exons, which code almost exclusively for signal peptides in each case. The first intron of GP1G does contain a region with high similarity to the coding exon of a human seminal vesicle secretory protein gene, semenogelin II. The 3' half of the GP1G gene appears to share a common ancestry with the human SKALP/elafin gene. Sequences related to the elafin promoter, coding, untranslated regions, and introns are clearly identifiable within the GP1G sequence. The elafin gene codes for a serine protease inhibitor and is expressed in a variety of different human tissues. To determine if the GP1G gene was also active outside of the seminal vesicle, RNA from a variety of guinea pig tissues was hybridized to a GP1G cDNA probe. At least three novel RNA bands hybridizing to the GP1G probe were detected in testis RNA samples, and GP1G-related mRNAs were also found in other tissues. These data suggest that these seminal vesicle secretory proteins may have functional roles outside the reproductive system. more...
- Published
- 1996
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47. Thiopurine methyltransferase pharmacogenetics: human gene cloning and characterization of a common polymorphism.
- Author
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Szumlanski C, Otterness D, Her C, Lee D, Brandriff B, Kelsell D, Spurr N, Lennard L, Wieben E, and Weinshilboum R
- Subjects
- Amino Acid Sequence, Base Sequence, Cell Line, Chromosome Mapping, Chromosomes, Human, Pair 6, Cloning, Molecular, Humans, Molecular Sequence Data, Mutagenesis, Site-Directed, Methyltransferases genetics, Polymorphism, Genetic
- Abstract
Thiopurine methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. Individual variation in the toxicity and therapeutic efficacy of these drugs is associated with a common genetic polymorphism that controls levels of TPMT activity and immunoreactive protein in human tissues. Because of the clinical significance of the "pharmacogenetic" regulation of this enzyme, it would be important to clone the gene for TPMT in humans and to study the molecular basis for the genetic polymorphism. As a first step toward cloning the gene for TPMT, we used the rapid amplification of genomic DNA ends to obtain a TPMT-specific intron sequence. That DNA sequence was used to design primers for the polymerase chain reaction (PCR), which made it possible to determine that the active gene for TPMT is located on human chromosome 6. A TPMT-positive cosmid clone was then isolated from a human chromosome 6-specific genomic DNA library, and the gene was sublocalized to chromosome band 6p22.3 by fluorescence in situ hybridization. The gene for TPMT was found to be approximately 34 kb in length and consisted of 10 exons and 9 introns. On the basis of the results of 5'-rapid amplification of cDNA ends, transcription initiation occurred at or near a point 89 nucleotides upstream from the translation initiation codon of previously reported TPMT cDNAs. Once the structure of the TPMT gene had been determined, it was possible to perform the PCR with primers complementary to the sequences of introns flanking each exon that encodes enzyme protein with template DNA obtained from subjects with known phenotypes for the TPMT genetic polymorphism. This DNA was isolated from blood samples from 4 unrelated subjects with genetically low TPMT activity and 4 unrelated subjects with high TPMT activity. All subjects with low TPMT activity were homozygous for two point mutations--a G-->A transition at nucleotide 460 in exon 7 and an A-->G transition at nucleotide 719 in exon 10. Both mutations resulted in alterations in amino acid sequence, with Ala-154-->Thr and Tyr-240-->Cys, respectively. All DNA samples isolated from the blood of subjects with high TPMT activity contained "wild-type" sequence. Results obtained with these blood samples were confirmed when DNA from four human liver samples with high TPMT activity were found to have wild-type sequence at nucleotides 460 and 719, while three liver samples with intermediate enzyme activity (i.e., samples presumed to be heterozygous for the polymorphism) were heterozygous for the exon 7 and exon 10 mutations present in the blood samples of homozygous low subjects. Transient expression in COS-1 cells of TPMT expression constructs that contained both of the mutations in exons 7 and 10, as well as each independently, demonstrated that each mutation, as well as both together, resulted in decreased expression of TPMT enzymatic activity and immunoreactive protein. Molecular cloning and structural characterization of the TPMT gene as well as elucidation of the molecular basis for a common TPMT genetic polymorphism will help make it possible to develop DNA-based diagnostic tests for the polymorphism and to determine the mechanism by which it results in decreased expression of this important drug-metabolizing enzyme. more...
- Published
- 1996
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48. Inhibitors of renal epithelial phosphate transport in tumor-induced osteomalacia and uremia.
- Author
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Kumar R, Haugen JD, Wieben ED, Londowski JM, and Cai Q
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Biological Transport drug effects, Cells, Cultured, Cross Reactions, Cyclic AMP metabolism, Epithelium metabolism, Female, Hemangioma complications, Hemangioma genetics, Humans, Kidney Failure, Chronic metabolism, Molecular Sequence Data, Neoplasm Proteins genetics, Neoplasm Proteins immunology, Opossums, Osteomalacia etiology, Parathyroid Hormone immunology, Phosphates antagonists & inhibitors, Recombinant Proteins pharmacology, Sequence Analysis, DNA, Sodium metabolism, Uremia etiology, Hemangioma metabolism, Kidney metabolism, Neoplasm Proteins pharmacology, Osteomalacia metabolism, Phosphates metabolism, Uremia metabolism
- Abstract
Tumors such as sclerosing hemangiomas are sometimes associated with hypophosphatemia and osteomalacia, both of which disappear on removal of the tumor. We identified a heat labile, 8,000-25,000 dalton, inhibitor of renal epithelial phosphate transport in supernatants of cultured sclerosing hemangioma cells obtained from a patient with oncogenic osteomalacia and hypophosphatemia. The inhibitor does not alter glucose or alanine transport in renal epithelial cells, and has a mechanism of cellular action distinct from that of parathyroid hormone (PTH) in that it inhibits phosphate transport in renal epithelia without increasing concentrations of cyclic 3',5' adenosine monophosphate (cAMP); it's activity is not blocked by a PTH receptor antagonist. Sclerosing hemangioma cells also produce a material that cross-reacts with antisera directed against PTH and tumor tissue sections immunostain with PTH antibodies. We have characterized a cDNA that encodes the PTH immunoreactive material. In its longest open reading frame the cDNA encodes a protein of 381 amino acids that does not resemble PTH in its primary structure. Opossum kidney cells transfected with the cDNA do not produce a product that inhibits phosphate transport. Dialysates from patients with end-stage renal disease also contain a substance(s) that inhibits phosphate and glucose transport in opossum kidney cells. The inhibitor(s) of phosphate uptake in dialysates is a heat labile, approximately 30,000 dalton substance that inhibits phosphate transport by a cAMP-independent mechanism. Determination of the structures and physiology of these phosphate transport inhibitors is likely to yield insights into the control of phosphate homeostasis. more...
- Published
- 1995
49. Abnormal processing of the human cholecystokinin receptor gene in association with gallstones and obesity.
- Author
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Miller LJ, Holicky EL, Ulrich CD, and Wieben ED
- Subjects
- Adult, Base Sequence, Cholesterol metabolism, Consensus Sequence, DNA, Complementary, Exons, Female, Humans, Molecular Sequence Data, Receptors, Cholecystokinin metabolism, Cholelithiasis genetics, Obesity genetics, Receptors, Cholecystokinin genetics
- Abstract
Background & Aims: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product., Methods: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient., Results: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient., Conclusions: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients. more...
- Published
- 1995
- Full Text
- View/download PDF
50. Androgen regulation of an elastase-like protease activity in the seminal vesicle.
- Author
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Harvey S, Vrabel A, Smith S, and Wieben E
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Chromogenic Compounds metabolism, Epithelium drug effects, Epithelium enzymology, Guinea Pigs, Isoflurophate pharmacology, Kinetics, Male, Molecular Sequence Data, Oligopeptides metabolism, Orchiectomy, Seminal Vesicles drug effects, Testosterone pharmacology, Androgens pharmacology, Pancreatic Elastase metabolism, Seminal Vesicles enzymology
- Abstract
The processing of secretory proteins in the guinea pig (GP) seminal vesicle epithelium (SVE) is altered by castration and restored by treatment of animals with androgens. To test the hypothesis that the changes in protein processing are due to changes in the activity of specific proteases, we examined the GPSVE for protease activities capable of cleaving a synthetic elastase substrate, succinyl-alanyl-alanyl-alanyl-p-nitroanilide (Suc(Ala)3pNA). We found that the GPSVE does contain a Suc(Ala)3pNA-cleaving activity that is sensitive to the serine protease inhibitor diisopropylfluorophosphate (DFP) and to the elastase inhibitor elastatinal. Furthermore, the amount of protease activity per milligram of SVE protein is reduced to about 50% of control levels by castration. The activity is completely restored within four days by treatment of castrated animals with androgens, but is not restored by treatment with estradiol, progesterone, or dexamethasone. Although the SVE enzyme did not yield a pattern of specific cleavage products when incubated with a secretory protein substrate in vitro, this enzyme activity was competitively inhibited by a peptide whose primary sequence included the cleavage site used by the processing machinery in vivo. more...
- Published
- 1995
- Full Text
- View/download PDF
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