Home About us Contact
|
Home About us Contact | ||
|
|
||
Longer Sequences (longer + sequence)
Selected AbstractsIntegrin ,3,1 interacts with I1PP2A/lanp and phosphatase PP1JOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Diana Mutz Abstract Integrin ,3,1 is a receptor for the extracellular matrix component laminin 5. To elucidate possible signaling pathways induced by integrin ,3,1, we looked for proteins that interact with the cytoplasmic part of the ,3A integrin subunit. We identified several multifunctional proteins by affinity chromatography and subsequent MALDI-TOF-MS and focused on the inhibitor 1 of serine/threonine phosphatase PP2A (I1PP2A, synonym: lanp) which also plays a role during the development of the mouse cerebellum. I1PP2A/lanp colocalizes with the ,3A integrin subunit in differentiated PC12 cells in the cell body and in neurites as well as in Purkinje cells of mouse cerebellum. Overexpression of GFP-I1PP2A/lanp in PC12 cells leads to markedly reduced neurite length on laminin 5 after induction with nerve growth factor. By affinity chromatography the protein phosphatase PP1 can also be identified as a ,3A/cyto-binding protein. PP1 and integrin ,3,1 can be pulled down by GST-I1PP2A/lanp from cell lysates of differentiated and undifferentiated PC12 cells. The phosphatase binds to the cytoplasmic membrane-proximal conserved GFFKR motif of the , integrin subunit, whereas I1PP2A/lanp requires a longer sequence for binding. PP1 but not PP2A is able to dephosphorylate precipitated integrin ,3,1 in vitro. Furthermore, PP1 releases phosphate from T1046 of phosphopeptides that mimic the phosphorylation consensus sequence in the cytoplasmic part of the ,3A integrin subunit. These data suggest that I1PP2A/lanp forms a complex with PP1 and the ,3A integrin subunit and might possibly regulate the phosphorylation status of integrin ,3,1 and/or integrin downstream targets. © 2006 Wiley-Liss, Inc. [source] DNA barcoding of stylommatophoran land snails: a test of existing sequencesMOLECULAR ECOLOGY RESOURCES, Issue 4 2009ANGUS DAVISON Abstract DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour-joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny-based methods for barcoding. There was, however, a large overlap between intra- and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2-parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone. [source] Labeling G-rich oligonucleotides (GROs) with N-succinimidyl 4-[18F]fluorobenzoate (S18FB)JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 14 2006Junling Li Abstract Three G-rich oligonucleotides (GROs) were conjugated with N-succinimidyl 4-[18F]fluorobenzoate (S18FB). The yields for GRO5, the shortest sequence among those three GROs being tested, were 42, 70, 84, and 99% at GRO5 concentration of 59, 118, 236, and 472 nmol/100 µl, respectively. Reaction temperature (22 and 40°C), reaction time (15 and 30 min), and borate buffer concentration (25 and 50 mM) showed no noticeable effects on the labeling results. Two longer sequences of GROs, GRO15A and GRO26A, were also labeled with the same procedures and the corresponding yields were 80 ± 2% at 93 nmol/100 µl (n = 3) and 47 ± 3% at 98 nmol/100 µl (n = 3), respectively. Finally, CROa was conjugated with S18FB to further confirm our results. The radiochemical yields were 13.7 ± 2.5, 40.9 ± 3.5, 70.1 ± 2.0, 93.4 ± 1.4% at 8.7, 17.5, 35 and 52.5 OD of CROa (non-isolated, decay-corrected and based on S18FB, n = 3) in comparison to 7, 17, 22 and 28% at 5, 10, 20 and 40 OD of CROa, respectively, as reported by Hedberg et al. (Acta Chem Scand 1998; 52: 1034,1039). Copyright © 2006 John Wiley & Sons, Ltd. [source] Temperature dependence and resonance assignment of 13C NMR spectra of selectively and uniformly labeled fusion peptides associated with membranesMAGNETIC RESONANCE IN CHEMISTRY, Issue 2 2004Michele L. Bodner Abstract HIV-1 and influenza viral fusion peptides are biologically relevant model fusion systems and, in this study, their membrane-associated structures were probed by solid-state NMR 13C chemical shift measurements. The influenza peptide IFP-L2CF3N contained a 13C carbonyl label at Leu-2 and a 15N label at Phe-3 while the HIV-1 peptide HFP-UF8L9G10 was uniformly 13C and 15N labeled at Phe-8, Leu-9 and Gly-10. The membrane composition of the IFP-L2CF3N sample was POPC,POPG (4:1) and the membrane composition of the HFP-UF8L9G10 sample was a mixture of lipids and cholesterol which approximately reflects the lipid headgroup and cholesterol composition of host cells of the HIV-1 virus. In one-dimensional magic angle spinning spectra, labeled backbone 13C were selectively observed using a REDOR filter of the 13C,15N dipolar coupling. Backbone chemical shifts were very similar at ,50 and 20°C, which suggests that low temperature does not appreciably change the peptide structure. Relative to ,50°C, the 20°C spectra had narrower signals with lower integrated intensity, which is consistent with greater motion at the higher temperature. The Leu-2 chemical shift in the IFP-L2CF3N sample correlates with a helical structure at this residue and is consistent with detection of helical structure by other biophysical techniques. Two-dimensional 13C,13C correlation spectra were obtained for the HFP-UF8L9G10 sample and were used to assign the chemical shifts of all of the 13C labels in the peptide. Secondary shift analysis was consistent with a ,-strand structure over these three residues. The high signal-to-noise ratio of the 2D spectra suggests that membrane-associated fusion peptides with longer sequences of labeled amino acids can also be assigned with 2D and 3D methods. Copyright © 2004 John Wiley & Sons, Ltd. [source] DNA barcoding of stylommatophoran land snails: a test of existing sequencesMOLECULAR ECOLOGY RESOURCES, Issue 4 2009ANGUS DAVISON Abstract DNA barcoding has attracted attention because it is a potentially simple and universal method for taxonomic assignment. One anticipated problem in applying the method to stylommatophoran land snails is that they frequently exhibit extreme divergence of mitochondrial DNA sequences, sometimes reaching 30% within species. We therefore trialled the utility of barcodes in identifying land snails, by analysing the stylommatophoran cytochrome oxidase subunit I sequences from GenBank. Two alignments of 381 and 228 base pairs were used to determine potential error rates among a test data set of 97 or 127 species, respectively. Identification success rates using neighbour-joining phylogenies were 92% for the longer sequence and 82% for the shorter sequence, indicating that a high degree of mitochondrial variation may actually be an advantage when using phylogeny-based methods for barcoding. There was, however, a large overlap between intra- and interspecific variation, with assignment failure (per cent of samples not placed with correct species) particularly associated with a low degree of mitochondrial variation (Kimura 2-parameter distance < 0.05) and a small GenBank sample size (< 25 per species). Thus, while the optimum intra/interspecific threshold value was 4%, this was associated with an overall error of 32% for the longer sequences and 44% for the shorter sequences. The high error rate necessitates that barcoding of land snails is a potentially useful method to discriminate species of land snail, but only when a baseline has first been established using conventional taxonomy and sample DNA sequences. There is no evidence for a barcoding gap, ruling out species discovery based on a threshold value alone. [source] | |||||||||||||