| 1. |
- Bolmeson, Caroline, et al.
(författare)
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Differences in islet-enriched miRNAs in healthy and glucose intolerant human subjects.
- 2011
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; Dec, s. 16-22
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Tidskriftsartikel (refereegranskat)abstract
- Many microRNAs (miRNAs) are known to be cell-type specific and are implicated in development of diseases. We investigated the global expression pattern of miRNAs in human pancreatic islets compared to liver and skeletal muscle, using bead-based technology and quantitative RT-PCR. In addition to the known islet-specific miR-375, we also found enrichment of miR-127-3p, miR-184, miR-195 and miR-493∗ in the pancreatic islets. The expression of miR-375, miR-127-3p, miR-184 and the liver-enriched miR-122 were positively correlated to insulin biosynthesis, while the expression of miR-127-3p and miR-184 were negatively correlated to glucose-stimulated insulin secretion (GSIS). These correlations were absent in islets of glucose intolerant donors (HbA1c⩾6.1). We suggest the presence of an islet-specific miRNA network, which consists of at least miR-375, miR-127-3p and miR-184, potentially involved in insulin secretion. Our results provide new insight into miRNA-mediated regulation of insulin secretion in healthy and glucose intolerant subjects.
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| 2. |
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| 3. |
- Carlsson, Karin, et al.
(författare)
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Effects on the conformation of FVIIa by sTF and Ca(2+) binding : Studies of fluorescence resonance energy transfer and quenching
- 2011
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 413:4, s. 545-549
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Tidskriftsartikel (refereegranskat)abstract
- The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 1044-1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF. Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.
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| 4. |
- Cedernaes, Jonathan, et al.
(författare)
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Comprehensive analysis of localization of 78 solute carrier genes throughout the subsections of the rat gastrointestinal tract
- 2011
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 411:4, s. 702-707
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Tidskriftsartikel (refereegranskat)abstract
- Solute carriers (SLCs), the second largest super-family of membrane proteins in the human genome, transport amino acids, sugars, fatty acids, inorganic ions, essential metals and drugs over membranes. To date no study has provided a comprehensive analysis of SLC localization along the entire GI tract. The aim of the present study was to provide a comprehensive, segment-specific description of the localization of SLC genes along the rat Cl tract by employing bioinformatics and molecular biology methods. The Unigene database was screened for rat SLC entries in the intestinal tissue. Using qPCR we measured expression of the annotated genes in the Cl tract divided into the following segments: the esophagus, the corpus and the antrum of the stomach, the proximal and distal parts of the duodenum, ileum, jejunum and colon, and the cecum. Our Unigene-derived gene pool was expanded with data from in-house tissue panels and a literature search. We found 44 out of 78 (56%) of gut SLC transcripts to be expressed in all Cl tract segments, whereas the majority of remaining SLCs were detected in more than five segments. SLCs are predominantly expressed in gut regions with absorptive functions although expression was also found in segments unrelated to absorption. The proximal jejunum had the highest number of differentially expressed SLCs. In conclusion, SLCs are a crucial molecular component of the Cl tract, with many of them expressed along the entire GI tract. This work presents the first overall road map of localization of transporter genes in the Cl tract.
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| 5. |
- del Pino, Isabel, et al.
(författare)
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The trafficking proteins Vacuolar Protein Sorting 35 and Neurobeachin interact with the glycine receptor beta-subunit
- 2011
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 412:3, s. 435-440
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Tidskriftsartikel (refereegranskat)abstract
- Inhibitory glycine receptors (GlyRs) are densely packed in the postsynaptic membrane due to a high-affinity interaction of their beta-subunits with the scaffolding protein gephyrin. Here, we used an affinity-based proteomic approach to identify the trafficking proteins Vacuolar Protein Sorting 35 (Vps35) and Neurobeachin (Nbea) as novel GlyR beta-subunit (GlyR beta) interacting proteins in rat brain. Recombinant Vps35 and a central fragment of Nbea bound to the large intracellular loop of GlyR beta in glutathione-S-transferase pull-downs; in addition, Vps35 displayed binding to gephyrin. Immunocytochemical staining of spinal cord sections revealed Nbea immunoreactivity apposed to and colocalizing with marker proteins of inhibitory synapses. Our data are consistent with roles of Vps35 and Nbea in the retrieval and post-Golgi trafficking of synaptic GlyRs and possibly other neurotransmitter receptors.
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| 6. |
- Deng, Hua, et al.
(författare)
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Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9
- 2011
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier BV. - 0006-291X .- 1090-2104. ; 404:2, s. 667-671
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Tidskriftsartikel (refereegranskat)abstract
- The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the beta-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7-higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.
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| 7. |
- Djerf, Emelie, et al.
(författare)
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The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo
- 2011
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 414:3, s. 563-568
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Tidskriftsartikel (refereegranskat)abstract
- The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC(50)) was approximately 0.8 mu M and by 5 mu M both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 mu M canertinib accumulated the cells in the G(1)-phase of the cell cycle within 24 h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 mu M canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses less than= 5 mu M canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.
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