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author:("Borup, hannah")
1.  IGF-I, IGF-II, and Insulin Stimulate Different Gene Expression Responses through Binding to the IGF-I Receptor 
Insulin and the insulin-like growth factors (IGF)-I and -II are closely related peptides important for regulation of metabolism, growth, differentiation, and development. The IGFs exert their main effects through the IGF-I receptor. Although the insulin receptor is the main physiological receptor for insulin, this peptide hormone can also bind at higher concentrations to the IGF-I receptor and exert effects through it. We used microarray gene expression profiling to investigate the gene expression regulated by IGF-I, IGF-II, and insulin after stimulation of the IGF-I receptor. Fibroblasts from mice, knockout for IGF-II and the IGF-II/cation-independent mannose-6-phosphate receptor, and expressing functional IGF-I but no insulin receptors, were stimulated for 4 h with equipotent saturating concentrations of insulin, IGF-I, and IGF-II. Each ligand specifically regulated a group of transcripts that was not regulated by the other two ligands. Many of the functions and pathways these regulated genes were involved in, were consistent with the known biological effects of these ligands. The differences in gene expression might therefore account for some of the different biological effects of insulin, IGF-I, and IGF-II. This work adds to the evidence that not only the affinity of a ligand determines its biological response, but also its nature, even through the same receptor.
doi:10.3389/fendo.2013.00098
PMCID: PMC3738877  PMID: 23950756
IGF-I receptor; microarray gene expression; insulin; IGF; differential signaling
2.  Plasticity of Adult Human Pancreatic Duct Cells by Neurogenin3-Mediated Reprogramming 
PLoS ONE  2012;7(5):e37055.
Aims/Hypothesis
Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it.
Methods
The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming.
Results
Ngn3 stimulates duct cells to express a focused set of genes that are characteristic for islet endocrine cells and/or neural tissues. This neuro-endocrine shift however, is incomplete with less than 10% of full duct-to-endocrine reprogramming achieved. Transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1.
Conclusions/Interpretation
The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes.
doi:10.1371/journal.pone.0037055
PMCID: PMC3351393  PMID: 22606327
3.  UPF2 Is a Critical Regulator of Liver Development, Function and Regeneration 
PLoS ONE  2010;5(7):e11650.
Background
Nonsense-mediated mRNA decay (NMD) is a post-transcriptional RNA surveillance process that facilitates the recognition and destruction of mRNAs bearing premature terminations codons (PTCs). Such PTC-containing (PTC+) mRNAs may arise from different processes, including erroneous processing and expression of pseudogenes, but also from more regulated events such as alternative splicing coupled NMD (AS-NMD). Thus, the NMD pathway serves both as a silencer of genomic noise and a regulator of gene expression. Given the early embryonic lethality in NMD deficient mice, uncovering the full regulatory potential of the NMD pathway in mammals will require the functional assessment of NMD in different tissues.
Methodology/Principal Findings
Here we use mouse genetics to address the role of UPF2, a core NMD component, in the development, function and regeneration of the liver. We find that loss of NMD during fetal liver development is incompatible with postnatal life due to failure of terminal differentiation. Moreover, deletion of Upf2 in the adult liver results in hepatosteatosis and disruption of liver homeostasis. Finally, NMD was found to be absolutely required for liver regeneration.
Conclusion/Significance
Collectively, our data demonstrate the critical role of the NMD pathway in liver development, function and regeneration and highlights the importance of NMD for mammalian biology.
doi:10.1371/journal.pone.0011650
PMCID: PMC2906512  PMID: 20657840
4.  Gene Expression Profiling of Placentas Affected by Pre-Eclampsia 
Several studies point to the placenta as the primary cause of pre-eclampsia. Our objective was to identify placental genes that may contribute to the development of pre-eclampsia. RNA was purified from tissue biopsies from eleven pre-eclamptic placentas and eighteen normal controls. Messenger RNA expression from pooled samples was analysed by microarrays. Verification of the expression of selected genes was performed using real-time PCR. A surprisingly low number of genes (21 out of 15,000) were identified as differentially expressed. Among these were genes not previously associated with pre-eclampsia as bradykinin B1 receptor and a 14-3-3 protein, but also genes that have already been connected with pre-eclampsia, for example, inhibin beta A subunit and leptin. A low number of genes were repeatedly identified as differentially expressed, because they may represent the endpoint of a cascade of events effectuated throughout gestation. They were associated with transcriptional regulation and vasoregulative pathways, along with a number of hypothetical proteins and gene sequences with unknown functions.
doi:10.1155/2010/787545
PMCID: PMC2831461  PMID: 20204130
5.  Gene Expression in Skeletal Muscle Biopsies from People with Type 2 Diabetes and Relatives: Differential Regulation of Insulin Signaling Pathways 
PLoS ONE  2009;4(8):e6575.
Background
Gene expression alterations have previously been associated with type 2 diabetes, however whether these changes are primary causes or secondary effects of type 2 diabetes is not known. As healthy first degree relatives of people with type 2 diabetes have an increased risk of developing type 2 diabetes, they provide a good model in the search for primary causes of the disease.
Methods/Principal Findings
We determined gene expression profiles in skeletal muscle biopsies from Caucasian males with type 2 diabetes, healthy first degree relatives, and healthy controls. Gene expression was measured using Affymetrix Human Genome U133 Plus 2.0 Arrays covering the entire human genome. These arrays have not previously been used for this type of study. We show for the first time that genes involved in insulin signaling are significantly upregulated in first degree relatives and significantly downregulated in people with type 2 diabetes. On the individual gene level, 11 genes showed altered expression levels in first degree relatives compared to controls, among others KIF1B and GDF8 (myostatin). LDHB was found to have a decreased expression in both groups compared to controls.
Conclusions/Significance
We hypothesize that increased expression of insulin signaling molecules in first degree relatives of people with type 2 diabetes, work in concert with increased levels of insulin as a compensatory mechanism, counter-acting otherwise reduced insulin signaling activity, protecting these individuals from severe insulin resistance. This compensation is lost in people with type 2 diabetes where expression of insulin signaling molecules is reduced.
doi:10.1371/journal.pone.0006575
PMCID: PMC2719801  PMID: 19668377
6.  Expression analyses of human cleft palate tissue suggest a role for osteopontin and immune related factors in palatal development 
Cleft lip and/or palate (CL/P) is a common congenital malformation with a complex etiology which is not fully elucidated yet. Epidemiological studies point to different etiologies in the cleft lip and palate subgroups, isolated cleft lip (CL), isolated cleft palate (CP) and combined cleft lip and palate (CLP). In order to understand the biological basis in these cleft lip and palate subgroups better we studied the expression profiles in human tissue from patients with CL/P. In each of the CL/P subgroups, samples were obtained from three patients and gene expression analysis was performed. Moreover, selected differentially expressed genes were analyzed by quantitative RT-PCR, and by immunohistochemical staining of craniofacial tissue from human embryos. Osteopontin (SPP1) and other immune related genes were significantly higher expressed in palate tissue from patients with CLP compared to CP and immunostaining in palatal shelves against SPP1, chemokine receptor 4 (CXCR4) and serglycin (PRG1) in human embryonic craniofacial tissue were positive, supporting a role for these genes in palatal development. However, gene expression profiles are subject to variations during growth and therefore we recommend that future gene expression in CL/P studies should use tissue from the correct embryonic time and place if possible, to overcome the biases in the presented study.
doi:10.3858/emm.2009.41.2.010
PMCID: PMC2679333  PMID: 19287188
cleft lip and palate; congenital abnormalities; gene expression profiling; osteopontin; serglycin
7.  Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs 
BMC Bioinformatics  2002;3:33.
Background
We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip) containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences). This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene), with each probe evaluated for hybridization kinetics (Tm) and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray.
Results
Hybridization of human muscle cRNAs to this MuscleChip (33 samples) showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41%) were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39%) could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms.
Conclusion
Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex) in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.
doi:10.1186/1471-2105-3-33
PMCID: PMC137597  PMID: 12456269
Expression profiling; oligonucleotide microarrays; Affymetrix; muscle; EST
8.  Sources of variability and effect of experimental approach on expression profiling data interpretation 
BMC Bioinformatics  2002;3:4.
Background
We provide a systematic study of the sources of variability in expression profiling data using 56 RNAs isolated from human muscle biopsies (34 Affymetrix MuscleChip arrays), and 36 murine cell culture and tissue RNAs (42 Affymetrix U74Av2 arrays).
Results
We studied muscle biopsies from 28 human subjects as well as murine myogenic cell cultures, muscle, and spleens. Human MuscleChip arrays (4,601 probe sets) and murine U74Av2 Affymetrix microarrays were used for expression profiling. RNAs were profiled both singly, and as mixed groups. Variables studied included tissue heterogeneity, cRNA probe production, patient diagnosis, and GeneChip hybridizations. We found that the greatest source of variability was often different regions of the same patient muscle biopsy, reflecting variation in cell type content even in a relatively homogeneous tissue such as muscle. Inter-patient variation was also very high (SNP noise). Experimental variation (RNA, cDNA, cRNA, or GeneChip) was minor. Pre-profile mixing of patient cRNA samples effectively normalized both intra- and inter-patient sources of variation, while retaining a high degree of specificity of the individual profiles (86% of statistically significant differences detected by absolute analysis; and 85% by a 4-pairwise comparison survival method).
Conclusions
Using unsupervised cluster analysis and correlation coefficients of 92 RNA samples on 76 oligonucleotide microarrays, we found that experimental error was not a significant source of unwanted variability in expression profiling experiments. Major sources of variability were from use of small tissue biopsies, particularly in humans where there is substantial inter-patient variability (SNP noise).
doi:10.1186/1471-2105-3-4
PMCID: PMC65691  PMID: 11936955
9.  Expression Profiling in the Muscular Dystrophies 
The Journal of Cell Biology  2000;151(6):1321-1336.
We used expression profiling to define the pathophysiological cascades involved in the progression of two muscular dystrophies with known primary biochemical defects, dystrophin deficiency (Duchenne muscular dystrophy) and α-sarcoglycan deficiency (a dystrophin-associated protein). We employed a novel protocol for expression profiling in human tissues using mixed samples of multiple patients and iterative comparisons of duplicate datasets. We found evidence for both incomplete differentiation of patient muscle, and for dedifferentiation of myofibers to alternative lineages with advancing age. One developmentally regulated gene characterized in detail, α-cardiac actin, showed abnormal persistent expression after birth in 60% of Duchenne dystrophy myofibers. The majority of myofibers (∼80%) remained strongly positive for this protein throughout the course of the disease. Other developmentally regulated genes that showed widespread overexpression in these muscular dystrophies included embryonic myosin heavy chain, versican, acetylcholine receptor α-1, secreted protein, acidic and rich in cysteine/osteonectin, and thrombospondin 4. We hypothesize that the abnormal Ca2+ influx in dystrophin- and α-sarcoglycan–deficient myofibers leads to altered developmental programming of developing and regenerating myofibers. The finding of upregulation of HLA-DR and factor XIIIa led to the novel identification of activated dendritic cell infiltration in dystrophic muscle; these cells mediate immune responses and likely induce microenvironmental changes in muscle. We also document a general metabolic crisis in dystrophic muscle, with large scale downregulation of nuclear-encoded mitochondrial gene expression. Finally, our expression profiling results show that primary genetic defects can be identified by a reduction in the corresponding RNA.
PMCID: PMC2190600  PMID: 11121445
muscular dystrophy; microarray; dystrophin; α-sarcoglycan; expression profiling

Results 1-9 (9)