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81.
Jeroen C. Verheijen Ker Yu Lourdes Toral-Barza Irwin Hollander Arie Zask 《Bioorganic & medicinal chemistry letters》2010,20(1):375-379
2-Aryl-4-morpholinothieno[3,2-d]pyrimidines are known PI3K inhibitors. This class of compounds also potently inhibited the homologous enzyme mTOR. Replacement of the morpholine group in these compounds with an 8-oxa-3-azabicyclo[3.2.1]octane group led to mTOR inhibitors with selectivity over PI3K. Optimization of the 2-aryl substituent led to the discovery of 2-(4-ureidophenyl)-thienopyrimidines as highly potent (IC50 <1 nM) mTOR inhibitors with excellent selectivity (up to >1000-fold) over PI3K and good potency in a cellular proliferation assay (IC50 <50 nM). 相似文献
82.
Verberckmoes S Van Soom A De Pauw I Dewulf J Vervaet C de Kruif A 《Theriogenology》2004,61(1):103-115
A new artificial insemination device for semen deposition near the utero-tubal junction in cattle (Ghent device) has been developed at the Ghent University (Belgium). In this study, the effect of the new insemination device on sperm quality was evaluated. Moreover, in a field trial 4064 dairy cows were inseminated by 12 inseminators to examine the efficacy of the device under field conditions.The Ghent device is a disposable plastic catheter which can easily follow the curvature of the uterine horns and thus reach the utero-tubal junction (UTJ). After expulsion of the inseminate with 0.7 or 1.7 ml of air, 19.0% of the insemination dose remained in the insemination catheter. Sperm loss can be diminished to 9.0% of the original insemination dose when the insemination catheter is flushed with 0.1 ml of air, followed by 0.6 ml of physiological saline solution. No toxic effect of the insemination catheter on sperm quality or fertilizing capacity was found. In the field trial, sperm were inseminated in dairy cattle which were divided in three groups. The first group was inseminated in the uterine body with the conventional insemination device, the second group in the uterine body with the Ghent device, and the third group in the tip of both uterine horns with the Ghent device. Each insemination was performed with 10 x 10(6) to 15 x 10(6) frozen-thawed spermatozoa. The pregnancy rates (PRs) were significantly affected by the insemination technique (P = 0.02), by the inseminator (P = 0.01), by heifer or cow (P < 0.01), and by the insemination number (P < 0.01). Pregnancy rates obtained with the conventional insemination device (57.6%) were significantly better than those obtained with the Ghent device in the uterine body (52.7%) (P < 0.01), but did not differ significantly from those obtained after deep insemination into both uterine horns (53.8%) (P = 0.27). It can be concluded that the Ghent device is suitable for utero-tubal junction insemination of dairy cattle under field conditions. Whether the Ghent device is also suitable for insemination with lower insemination doses is at present under investigation. 相似文献
83.
Paleoclimatic history and vicariant speciation in the "sand goby" group (Gobiidae, Teleostei) 总被引:6,自引:0,他引:6
Vicariant and climatic cycling speciation hypotheses of the 'sand gobies' belonging to the genera Pomatoschistus, Gobiusculus, Knipowitschia, and Economidichthys are tested using molecular phylogenies constructed of nuclear DNA (ITS1 locus) and mitochondrial DNA (12S and 16S fragments). These gobies are among the most abundant in the Eastern Atlantic-Mediterranean region, and play an important role in the ecosystem. Considerable ITS1 length differences, primarily due to the presence of several tandem repeats, were found between species and even within individuals. Therefore, phylogenetic analyses focused on fragments of the 12S and 16S mtDNA region that have been sequenced for 16 goby taxa. The 'sand gobies' clustered as a monophyletic group as proposed on morphological grounds. However, G. flavescens, E. pygmaeus, and K. punctatissima clustered within the Pomatoschistus species, pointing to a paraphyletic origin of these genera. Furthermore, the genetic divergence between P. minutus from the Adriatic Sea versus the Atlantic-Mediterranean region was as high as the divergence within the P. minutus complex, suggesting that P. minutus from the Adriatic Sea should be considered as a distinct species. The "star" phylogeny might suggest that these gobies evolved in a very short time period, possibly linked to the drastic alterations in the Mediterranean Sea during and immediately after the Messinian salinity crisis at the end of the Miocene. The freshwater life-style appeared monophyletic; equating its origin with the salinity crisis resulted in a molecular clock estimate of 1.4% divergence per million years. The last common ancestor probably occupied sandy bottoms and a coastal niche while several species subsequently adapted to new habitats (pelagic, freshwater or stenohaline). The origin of the shallowest clades dated back to the glacial cycling during the Pleistocene epoch. 相似文献
84.
Houtenbos I Westers TM Stam AG de Gruijl TD Scheper RJ Ossenkoppele GJ van de Loosdrecht AA 《Cancer immunology, immunotherapy : CII》2003,52(7):455-462
PURPOSE: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore, we compared morphological, immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. METHODS: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days, respectively, in FCS-containing medium (FCS), StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. RESULTS: Serum-free culture of AML-APCs resulted in comparable morphology, relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. CONCLUSION: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological, immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP). 相似文献
85.
86.
87.
Identification of PLOD2 as telopeptide lysyl hydroxylase,an important enzyme in fibrosis 总被引:5,自引:0,他引:5
van der Slot AJ Zuurmond AM Bardoel AF Wijmenga C Pruijs HE Sillence DO Brinckmann J Abraham DJ Black CM Verzijl N DeGroot J Hanemaaijer R TeKoppele JM Huizinga TW Bank RA 《The Journal of biological chemistry》2003,278(42):40967-40972
The hallmark of fibrotic processes is an excessive accumulation of collagen. The deposited collagen shows an increase in pyridinoline cross-links, which are derived from hydroxylated lysine residues within the telopeptides. This change in cross-linking is related to irreversible accumulation of collagen in fibrotic tissues. The increase in pyridinoline cross-links is likely to be the result of increased activity of the enzyme responsible for the hydroxylation of the telopeptides (telopeptide lysyl hydroxylase, or TLH). Although the existence of TLH has been postulated, the gene encoding TLH has not been identified. By analyzing the genetic defect of Bruck syndrome, which is characterized by a pyridinoline deficiency in bone collagen, we found two missense mutations in exon 17 of PLOD2, thereby identifying PLOD2 as a putative TLH gene. Subsequently, we investigated fibroblasts derived from fibrotic skin of systemic sclerosis (SSc) patients and found that PLOD2 mRNA is highly increased indeed. Furthermore, increased pyridinoline cross-link levels were found in the matrix deposited by SSc fibroblasts, demonstrating a clear link between mRNA levels of the putative TLH gene (PLOD2) and the hydroxylation of lysine residues within the telopeptides. These data underscore the significance of PLOD2 in fibrotic processes. 相似文献
88.
Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity 总被引:2,自引:0,他引:2
Wingens M Walma T van Ingen H Stortelers C van Leeuwen JE van Zoelen EJ Vuister GW 《The Journal of biological chemistry》2003,278(40):39114-39123
Various chimeras of the ErbB1-specific ligands epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) display an enlarged repertoire as activators of ErbB2.ErbB3 heterodimers. Mutational analysis indicated that particularly residues in the N terminus and B-loop region of these ligands are involved in the broadened receptor specificity. In order to understand the receptor specificity of T1E, a chimeric ligand constructed by the introduction of the linear N-terminal region of TGFalpha into EGF, we determined in this study the solution structure and dynamics of T1E by multidimensional NMR analysis. Subsequently, we studied the structural characteristics of T1E binding to both ErbB1 and ErbB3 by superposition modeling of its structure on the known crystal structures of ErbB3 and liganded ErbB1 complexes. The results show that the overall structure of T1E in solution is very similar to that of native EGF and TGFalpha but that its N terminus shows an extended structure that is appropriately positioned to form a triple beta-sheet with the large antiparallel beta-sheet in the B-loop region. This conformational effect of the N terminus together with the large overall flexibility of T1E, as determined by 15N NMR relaxation analysis, may be a facilitative property for its broad receptor specificity. The structural superposition models indicate that hydrophobic and electrostatic interactions of the N terminus and B-loop of T1E are particularly important for its binding to ErbB3. 相似文献
89.
Scheper W Thaminy S Kais S Stagljar I Römisch K 《The Journal of biological chemistry》2003,278(39):37998-38003
Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins. 相似文献
90.
Bink RJ Habuchi H Lele Z Dolk E Joore J Rauch GJ Geisler R Wilson SW den Hertog J Kimata K Zivkovic D 《The Journal of biological chemistry》2003,278(33):31118-31127
Heparan sulfate proteoglycans function in development and disease. They consist of a core protein with attached heparan sulfate chains that are altered by a series of carbohydrate-modifying enzymes and sulfotransferases. Here, we report on the identification and characterization of a gene encoding zebrafish heparan sulfate 6-O-sulfotransferase (hs6st) that shows high homology to other heparan sulfate 6-O-sulfotransferases. When expressed as a fusion protein in cultured cells, the protein shows specific 6-O-sulfotransferase activity and preferentially acts on the iduronosyl N-sulfoglycosamine. In the developing embryo, hs6st is expressed in the brain, the somites, and the fins; the same structures that were affected upon morpholino-mediated functional knockdown. Morpholino injections significantly inhibited 6-O- but not 2-O-sulfation as assessed by HPLC. Morphants display disturbed somite specification independent of the somite oscillator mechanism and have impaired muscle differentiation. In conclusion, our results show that transfer of sulfate to specific positions on glycosaminoglycans is essential for muscle development. 相似文献