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791.
Vivian Chua Anna Han Nelisa Bechtel Timothy J. Purwin Emily Hunter Connie Liao J. William Harbour Andrew E. Aplin 《Pigment cell & melanoma research》2022,35(1):78-87
Metastatic uveal melanoma (UM) responds poorly to targeted therapies and immune checkpoint inhibitors. Loss of BRCA1-associated protein 1 (BAP1) via inactivating mutations in the BAP1 gene is associated with UM progression. Thus, molecular alterations caused by BAP1 dysfunction may be novel therapeutic targets for metastatic UM. Here, we found that phosphorylation of AMP-dependent kinase (AMPK) was elevated in BAP1-altered (or mutant) compared to BAP1-unaltered (or wild-type [WT]) UM tumors. As a readout of AMPK pathway activation, phosphorylation of an AMPK downstream effector, acetyl-CoA-carboxylase (ACC), was also elevated. BAP1 re-expression in BAP1-null UM cell lines decreased phospho-AMPK (pAMPK) and phospho-ACC (pACC) levels. AMPK phosphorylation is mediated by calcium/calmodulin dependent protein kinase kinase 2 (CaMKK2) and potentially liver kinase B1 (LKB1) in BAP1 mutant UM cells. Knockdown of AMPKα1/2 reduced the viability of BAP1 mutant UM cells, indicating a survival function of AMPK in BAP1 mutant UM. Our data suggest that the AMPK pathway is an important mechanism mediating the survival of BAP1 mutant UM. Targeting the AMPK pathway may be a novel therapeutic strategy for metastatic UM. 相似文献
792.
A comparison of amino acid sequences of yeast Rer2p and Srt1p Z-prenyltransferases shows that the spatial organization of their substrate tunnels agrees with that determined by X-ray for the E. coli undecaprenyl diphosphate synthase (UPPs). The observed trend in the maxima of product length distribution shifted from C(55) in UPPs to C(80) in Rer2p and to C(110) in Srt1p. This suggests a significant increase in the size of the enzyme hydrophobic tunnel from approximately 1000 A(3) of E. coli UPPs to approximately 1300 A(3) required to accommodate C(80) in Rer2p and to 1700 A(3) for C(110) in Srt1p. Moreover, Srt1p products reaching C(290) indicate the failure of a strict bacterial-like chain length control. On the basis of E. coli UPPs crystallographic structure the yeast Rer2p model was constructed. In the model three amino acid residues inserted into the sequence corresponding to the "floor" region of the tunnel extends the bottom loop what results in the required increase of the tunnel volume. Moreover, thermal fluctuations of this loop occasionally create a hole in the tunnel floor, making escape of polyprenol omega end out of the tunnel possible what switches off the control mechanism of product length thereby allowing a practically unlimited elongation process leading to an exponential distribution of longer chain polyprenols. 相似文献
793.
Wattanachaisaereekul S Lantz AE Nielsen ML Andrésson OS Nielsen J 《Biotechnology and bioengineering》2007,97(4):893-900
Polyketides are a group of natural products that have gained much interest due to their use as antibiotics, cholesterol lowering agents, immunosuppressors, and as other drugs. Many organisms that naturally produce polyketides are difficult to cultivate and only produce these metabolites in small amounts. It is therefore of general interest to transfer polyketide synthase (PKS) genes from their natural sources into heterologous hosts that can over-produce the corresponding polyketides. In this study we demonstrate the heterologous expression of 6-methylsalicylic acid synthase (6-MSAS), naturally produced by Penicillium patulum, in the yeast Saccharomyces cerevisiae. In order to activate the PKS a 4'-phosphopantetheinyl transferase (PPTase) is required. We therefore co-expressed PPTases encoded by either sfp from Bacillus subtilis or by npgA from Aspergillus nidulans. The different strains were grown in batch cultures. Growth and product concentration were measured and kinetic parameters were calculated. It was shown that both PPTases could be efficiently used for activation of PKS's in yeast as good yields of 6-MSA were obtained with both enzymes. 相似文献
794.
A stirred microchamber for oxygen consumption rate measurements with pancreatic islets 总被引:1,自引:0,他引:1
Improvements in pancreatic islet transplantation for treatment of diabetes are hindered by the absence of meaningful islet quality assessment methods. Oxygen consumption rate (OCR) has previously been used to assess the quality of organs and primary tissue for transplantation. In this study, we describe and characterize a stirred microchamber for measuring OCR with small quantities of islets. The device has a titanium body with a chamber volume of about 200 microL and is magnetically stirred and water jacketed for temperature control. Oxygen partial pressure (pO(2)) is measured by fluorescence quenching with a fiber optic probe, and OCR is determined from the linear decrease of pO(2) with time. We demonstrate that measurements can be made rapidly and with high precision. Measurements with betaTC3 cells and islets show that OCR is directly proportional to the number of viable cells in mixtures of live and dead cells and correlate linearly with membrane integrity measurements made with cells that have been cultured for 24 h under various stressful conditions. 相似文献
795.
A series of substituted 1-glucosyl-2-benzoylhydrazines were synthesized and their pseudo-first-order rate constants for hydrolysis were determined at pH 4, 5, 6 at 50 degrees C. All the compounds hydrolyzed quickly (t(1/2)<3h) at pH 4.0, but were increasingly stable as the pH approached neutrality. 相似文献
796.
The filamentous fungus, Aspergillus oryzae, was cultivated in batch and fed-batch cultivations in order to investigate the use of multi-wavelength fluorescence for monitoring course of events during filamentous fungi cultivations. The A. oryzae strain applied expressed a fungal lipase from Thermomyces lanuginosus. Spectra of multi-wavelength fluorescence were collected every 5 min with the BioView system (DELTA, Denmark) and both explorative and predictive models, correlating the fluorescence data with cell mass and lipase activity, were built. During the cultivations, A. oryzae displayed dispersed hyphal growth and under these conditions no fouling of the multi-wavelength fluorescence sensor was observed. The scores of a parallel factor analysis (PARAFAC) model, based on the fluorescence spectra, gave clear evidence of, for example, the on-set of the feeding phase. The predictive models, estimating the cell mass, showed correlations between 0.73 and 0.97 with root mean square error of cross validation (RMSECV) values between 1.48 and 0.77 g . kg(-1). A model estimating the lipase activity was also constructed for the fed-batch cultivations with a correlation of 0.93. The results presented here clearly show that multi-wavelength fluorescence is a useful tool for monitoring fed-batch cultivations of filamentous fungi. 相似文献
797.
Ortaldo JR Mason A Willette-Brown J Ruscetti FW Wine J Back T Stull T Bere EW Feigenbaum L Winkler-Pickett R Young HA 《Cellular immunology》2007,249(1):8-19
Analysis of the NK cell developmental pathway suggests that CD2 expression may be important in regulating NK maturation. To test this hypothesis, we developed mice containing only an inhibitory CD2 molecule by linking the extracellular domain of CD2 to an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM) motif. Mice containing the CD2 Tg(ITIM) transgene, introduced into a CD2 KO background, have no morphologically detectable lymph nodes, although development of the thymus appears normal. In addition, these mice had major loss of both NK and NKT subsets in peripheral organs, while T and B cell frequencies were intact. Expression of CD2 was low on T cells and lacking on B cells and functional defects were observed in these populations. NKT cells expressing CD4 were absent, while the CD8+ and double negative NKT cells were retained. Small subsets of NK cells were detected but expression of CD2 on these cells was very low or absent, and their maturation was impaired. Based on the phenotype described here, we believe that these mice represent a unique model to study lymphoid organ and lymphocyte development. 相似文献
798.
799.
Toth ZE Shahar T Leker R Szalayova I Bratincsák A Key S Lonyai A Németh K Mezey E 《Experimental cell research》2007,313(9):1943-1950
The green fluorescent protein (GFP) is among the most commonly used expression markers in biology. GFP-tagged cells have played a particularly important role in studies of cell lineage. Sensitive detection of GFP is crucially important for such studies to be successful, and problems with detection may account for discrepancies in the literature regarding the possible fate choices of stem cells. Here we describe a very sensitive technique for visualization of GFP. Using it we can detect about 90% of cells of donor origin while we could only see about 50% of these cells when we employ the methods that are in general use in other laboratories. In addition, we provide evidence that some cells permanently silence GFP expression. In the case of the progeny of bone marrow stem cells, it appears that the more distantly related they are to their precursors, the more likely it is that they will turn off the lineage marker. 相似文献
800.
Receptor for Activated C Kinase, RACK1, is an adaptor protein that regulates signaling via Src and PKC-dependent pathways, and has been implicated in cell migration. In this study we demonstrate novel functions for RACK1 in regulating adhesion dynamics during cell migration. We report that cells lacking RACK1 are less motile and show reduced dynamics of paxillin and talin at focal complexes. To investigate the role of the RACK1/Src interactions in adhesion dynamics, we used RACK1 in which the putative Src binding site has been mutated (RACK Y246F). RACK1-deficient cells showed enhanced c-Src activity that was rescued by expression of wild type RACK1, but not by RACK Y246F. Expression of wild type RACK1, but not RACK Y246F, was also able to rescue the adhesion and migration defects observed in the RACK1-deficient cells. Furthermore, our findings indicate that RACK1 functions to regulate paxillin phosphorylation and that its effects on paxillin dynamics require the Src-mediated phosphorylation of tyrosine 31/118 on paxillin. Taken together, these findings support a novel role for RACK1 as a key regulator of cell migration and adhesion dynamics through the regulation of Src activity, and the modulation of paxillin phosphorylation at early adhesions. 相似文献