Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report efficient methods for using functional proteomics to study signal transduction pathways in mouse fibroblasts following stimulation with PDGF. After stimulation, complete cellular proteins were separated using two-dimensional electrophoresis and phosphorylated proteins were detected with anti-phosphotyrosine and anti-phosphoserine antibodies. About 260 and 300 phosphorylated proteins were detected with the anti-phosphotyrosine and anti-phosphoserine antibodies, respectively, at least 100 of which showed prominent changes in phosphorylation as a function of time after stimulation. Proteins showing major time-dependent changes in phosphorylation were subjected to in-gel digestion with trypsin and identified by mass spectroscopy using MALDI-TOF mass fingerprinting and ESI peptide sequencing. We have observed phosphorylated proteins known to be part of the PDGF signal transduction pathway such as ERK 1, serine/threonine protein kinase akt and protein tyrosine phosphatase syp, proteins such as proto-oncogene tyrosine kinase fgr previously known to participate in other signal transduction pathways, and some proteins such as plexin-like protein with no previously known function in signal transduction. Information about the phosphorylation site was obtained for proto-oncogene tyrosine kinase fgr and for cardiac alpha-actin. The methods used here have proven to be suitable for the identification of time-dependent changes in large numbers of proteins involved in signal transduction pathways.
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PMID:Functional proteomics analysis of signal transduction pathways of the platelet-derived growth factor beta receptor. 1002 55

The gene Dtk, encoding the prohormone of tachykinin-related peptides (TRPs), has been identified from Drosophila. This gene encodes five putative tachykinin-related peptides (DTK-1 to 5) that share the C-terminal sequence FXGXRamide (where X represents variable residues) as well as an extended peptide (DTK-6) with the C-terminus FVAVRamide). By mass spectrometry (MALDI-TOF-MS), we identified ion signals with masses identical to those of DTK-1 to 5 in specific brain regions. We have analyzed the distribution of the Dtk transcript and peptides, by in situ hybridization and immunocytochemistry during postembryonic development of the central nervous system (CNS) of Drosophila. Antiserum against a cockroach TRP that cross-reacts with the DTKs was used for immunocytochemistry. Expression of transcript and peptides was detected from first to third instar larvae, through metamorphosis to adult flies. Throughout postembryonic development, we were able to follow the strong expression of TRPs in a pair of large descending neurons with cell bodies in the brain. The number of TRP-expressing neuronal cell bodies in the brain and ventral nerve cord increases during larval development. In the early pupa (stage P8), the number of TRP-expressing cell bodies is lower than in the third instar larvae. The number drastically increases during later pupal development, and in the adult fly about 200 TRP-expressing neurons can be seen in the CNS. The continuous expression of TRPs in neurons throughout postembryonic development suggests specific functional roles in both larval and imaginal flies and possibly also in some neurons during pupal development.
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PMID:Neuronal expression of tachykinin-related peptides and gene transcript during postembryonic development of Drosophila. 1289 11

From the Drosophila virilis late puff region 31C, we microcloned two neighbouring genes, Kil-1 and Kil-2, that encode putative Kunitz serine protease inhibitor like proteins. The Kil-1 gene is expressed exclusively in prepupal salivary glands. Using a size mutant of the KIL-1 protein and MALDI-TOF analysis, we demonstrate that during pupation this protein is released from the prepupal salivary glands into the pupation fluid covering the surface of the pupa. 3-D-structure predictions are consistent with the known crystal structure of the human Kunitz type protease inhibitor 2KNT. This is the first experimental proof for the extracorporal presence of a distinct Drosophila prepupal salivary gland protein. Possible functions of KIL-1 in the context of the control of proteolytic activities in the pupation fluid are discussed.
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PMID:A Kunitz type protease inhibitor related protein is synthesized in Drosophila prepupal salivary glands and released into the moulting fluid during pupation. 1526 89

Neural stem cells (NSCs) are multipotential progenitor cells that have self-renewal activity. Since the fates of the NSCs in situ depend on their niche containing growth factors and cytokines, we performed surface enhanced laser desorption/ionization time-of flight mass spectrometry (SELDI-TOF-MS) to screen for differentially secreted proteins in conditioned medium of neural stem cells and compared with that of NIH3T3 cells. A 15.3-kDa protein detected only in the conditioned medium of neural stem cells was determined as pleiotrophin (PTN) by SELDI-TOF-MS and ProteinChip-tandem MS systems. Identification of pleiotrophin was further confirmed by one-dimensional SDS gel electrophoresis and Edman degradation analysis. The mRNA transcripts of PTN and its receptors [receptor protein tyrosine phosphatase (RPTP) beta/zeta, N-syndecan and anaplastic lymphoma kinase (ALK)] were detected in neurosphere, suggesting that pleiotrophin signaling systems are present in the neural stem cells and are involved in the modulation of fate of neural stem cells.
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PMID:Identification of pleiotrophin in conditioned medium secreted from neural stem cells by SELDI-TOF and SELDI-tandem mass spectrometry. 1535 7

Chiral recognition is a fundamental phenomenon in life sciences, based on the enantioselective complexation of a chiral molecule with a chiral selector. The diastereomeric aggregates, formed by complexation, are held together by a different combination of intermolecular forces and are therefore endowed with different stability and reactivity. Determination of these forces, which are normally affected in the condensed phase by solvent and supramolecular interactions, requires the generation of the diastereomeric complexes in the isolated state and their spectroscopic investigation. This review deals with chiral recognition in the gas phase through the application of laser-resolved mass spectrometric techniques (R2PI-TOF and RET-MS). The measurement of the fragmentation thresholds of diastereomeric clusters by these techniques allows the determination of the nature of the intrinsic interactions, which control their formation and affect their stability and reactivity.
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PMID:Chiral recognition by mass-resolved laser spectroscopy. 1553 68

In this study, the frequency of BRAF mutation was investigated in a series of 67 cases of papillary thyroid cancer (PTC) in patients from Ukraine. Thirty-two patients were aged 30 years or older at the time of diagnosis and 35 were under 16. Tumour was microdissected from paraffin wax-embedded sections, DNA extracted, and the presence of the BRAF T1796A mutation demonstrated by two different methods: PCR followed by restriction enzyme digestion or primer extension assay and detection using MALDI-TOF mass spectrometry. Eighteen (58%) of the adult cases, but only one of the 35 cases aged less than 16 harboured a BRAF T1796A mutation. There was complete agreement between the two methods used, suggesting that the MALDI-TOF assay is a robust alternative to conventional mutation analysis. RET rearrangement was also examined in the young cohort. The overall frequency of RET rearrangement was 45.7%. Eight of the younger group of patients were born after 1 December 1986 and were therefore not exposed to radioiodine in fallout from Chernobyl. None of the PTCs from these eight patients were positive for BRAF mutation. The frequency of RET rearrangement was 44% in the 27 cases exposed to radiation and 50% in the eight not exposed. These results suggest that the different molecular biological profiles observed are associated with the age of the patient at diagnosis with PTC, rather than being associated with radiation exposure.
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PMID:Frequency of BRAF T1796A mutation in papillary thyroid carcinoma relates to age of patient at diagnosis and not to radiation exposure. 1571 93

ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interaction between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.
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PMID:Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease. 1597 76

We have designed resequencing microarrays to test the performance of this platform when interrogating a large number of exons (164 total) from genes associated with cancer. To evaluate false positive and negative rates, dideoxy sequencing was done for 335,420 bases interrogated by the arrays. From the array data, calls could be made for approximately 97.5% of the bases, and false positive rates were very low with only a single mutation reported from the array dataset for which the corresponding dideoxy trace had a clean wildtype sequence. For the nucleotide positions where array calls were made, false negative rates were 1.41% for heterozygous mutations. All the homozygous mutations were detected, but 8.11% were erroneously reported as heterozygous changes from the reference sequence by the array analysis software. In addition, 20 non-small cell lung cancer (NSCLC) samples were analyzed using the arrays, and both somatic and germline mutations were found. The most interesting findings were two MET mutations that have recently been implemented in NSCLC. Large scale MALDI-TOF genotyping indicated that one of these mutations (T1010I) might represent a true cancer-causing genotype, whereas the other (N375S) appears to be a common germline polymorphism.
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PMID:A transforming MET mutation discovered in non-small cell lung cancer using microarray-based resequencing. 1617 45

Most gastrointestinal stromal tumors (GIST) have activating mutations in either KIT or PDGFRA. However, a small subset of GIST lacks either mutation. To investigate the molecular characteristics of GIST according to mutation type, protein expression profiles in 12 GIST (2 cases with PDGFRA mutations, 8 cases with KIT mutations and 2 cases lacking either mutation) were analyzed using 2-DE and MALDI-TOF-MS. Comparative analysis of the respective spot patterns using 2-DE showed that 15 proteins were differently expressed according to the mutation status. Expression levels of septin and heat shock protein (HSP) 27 were increased in GIST with KIT mutations and annexin V was overexpressed in GIST lacking either mutation. Among the 15 proteins, overexpression of 5 proteins [annexin V, high mobility group protein 1 (HMGB1), C13orf2, glutamate dehydrogenase 1 and fibrinogen beta chain] and decreased expression of RoXaN correlated with a higher tumor grade. These findings suggest that differential protein expression can be used as a diagnostic biomarker. Moreover, it may play a role in the development and progression of GIST according to activating mutation type, as these proteins have been shown to be involved in tumor metastasis, apoptosis and immune response.
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PMID:Differentially expressed proteins in gastrointestinal stromal tumors with KIT and PDGFRA mutations. 1640 62

We report matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and off-line coupling of size-exclusion chromatography with MALDI-TOFMS analysis (SEC/MALDI-TOFMS) methods for the detailed characterization of poly[(R,S)-3-hydroxybutyrate-co-L-lactic acid], P[(R,S)-3HB-co-LA], and poly[(R,S)-3-hydroxybutyrate-co-epsilon-caprolactone], P[(R,S)-3HB-co-CL], copolymer samples which are expected to be used in special medical application as scaffolds for cartilage and soft tissue engineering. The novel copolyesters contained randomly distributed (R,S)-3-hydroxybutyrate structural units, were synthesized by transesterification of the corresponding homopolymers, i.e. atactic poly[(R,S)-3-hydroxybutyrate], a-PHB, and poly(L-Lactide) (PLLA) or poly(epsilon-caprolactone) (PCL), respectively. The MS methods used for the characterization of the resulting polydisperse copolyester samples were supported by classical methods (NMR, SEC). The structures of individual copolyester macromolecules, including end-group chemical structures, were established using initially MALDI-TOFMS and then SEC/MALDI-TOFMS. The compositions of the copolyesters were determined by two methods, namely based on 1H NMR and MALDI-TOF spectra. The two sets of values showed good agreement. The sequence distribution was determined using the signal intensities of individual copolyester macromolecules, which appeared in MALDI-TOF mass spectra. Furthermore, sequence analysis gave information about the degree of transesterification. The copolyesters synthesized, with only one exception, were demonstrated to be almost random, which implies that the ester-ester exchange was close to completion.
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PMID:Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with size-exclusion chromatographic fractionation for structural characterization of synthetic aliphatic copolyesters. 1647 Jul 27


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