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91.
92.
Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action 总被引:2,自引:0,他引:2
Zakrzewska M Krowarsch D Wiedlocha A Olsnes S Otlewski J 《Journal of molecular biology》2005,352(4):860-875
Fibroblast growth factor 1 (FGF-1) shows strong angiogenic, osteogenic and tissue-injury repair properties that might be relevant to medical applications. Since FGF-1 is partially unfolded at physiological temperature we decided to increase significantly its conformational stability and test how such an improvement will affect its biological function. Using an homology approach and rational strategy we designed two new single FGF-1 mutations: Q40P and S47I that appeared to be the most strongly stabilizing substitutions among those reported so far, increasing the denaturation temperature by 7.8 deg. C and 9.0 deg. C, respectively. As our goal was to produce highly stable variants of the growth factor, we combined these two mutations with five previously described stabilizing substitutions. The multiple mutants showed denaturation temperatures up to 27 deg. C higher than the wild-type and exhibited full additivity of the mutational effects. All those mutants were biologically competent in several cell culture assays, maintaining typical FGF-1 activities, such as binding to specific cell surface receptors and activation of downstream signaling pathways. Thus, we demonstrate that the low denaturation temperature of wild-type FGF-1 is not related to its fundamental cellular functions, and that FGF-1 action is not affected by its stability. A more detailed analysis of the biological behavior of stable FGF-1 mutants revealed that, compared with the wild-type, their mitogenic properties, as probed by the DNA synthesis assay, were significantly increased in the absence of heparin, and that their half-lives were extensively prolonged. We found that the biological action of the mutants was dictated by their susceptibility to proteases, which strongly correlated with the stability. Mutants which were much more resistant to proteolytic degradation always displayed a significant improvement in the half-life and mitogenesis. Our results show that engineered stable growth factor variants exhibit enhanced and prolonged activity, which can be advantageous in terms of the potential therapeutic applications of FGF-1. 相似文献
93.
Spassieva SD Rahmaniyan M Bielawski J Clarke CJ Kraveka JM Obeid LM 《Journal of lipid research》2012,53(5):918-928
We applied a metabolic approach to investigate the role of sphingolipids in cell density-induced growth arrest in neuroblastoma cells. Our data revealed that sphingolipid metabolism in neuroblastoma cells significantly differs depending on the cells' population context. At high cell density, cells exhibited G0/G1 cell-cycle arrest and reduced ceramide, monohexosylceramide, and sphingomyelin, whereas dihydroceramide was significantly increased. In addition, our metabolic-labeling experiments showed that neuroblastoma cells at high cell density preferentially synthesized very long chain (VLC) sphingolipids and dramatically decreased synthesis of sphingosine-1-phosphate (S1P). Moreover, densely populated neuroblastoma cells showed increased message levels of both anabolic and catabolic enzymes of the sphingolipid pathway. Notably, our metabolic-labeling experiments indicated reduced dihydroceramide desaturase activity at confluence, which was confirmed by direct measurement of dihydroceramide desaturase activity in situ and in vitro. Importantly, we could reduce dihydroceramide desaturase activity in low-density cells by applying conditional media from high-density cells, as well as by adding reducing agents, such as DTT and L-cysteine to the media. In conclusion, our data suggest a role of the sphingolipid pathway, dihydroceramides desaturase in particular, in confluence-induced growth arrest in neuroblastoma cells. 相似文献
94.
Tree species effects on coupled cycles of carbon, nitrogen, and acidity in mineral soils at a common garden experiment 总被引:4,自引:0,他引:4
Kevin E. Mueller David M. Eissenstat Sarah E. Hobbie Jacek Oleksyn Andrzej M. Jagodzinski Peter B. Reich Oliver A. Chadwick Jon Chorover 《Biogeochemistry》2012,111(1-3):601-614
Forest biogeochemical cycles are shaped by effects of dominant tree species on soils, but the underlying mechanisms are not well understood. We investigated effects of temperate tree species on interactions among carbon (C), nitrogen (N), and acidity in mineral soils from an experiment with replicated monocultures of 14 tree species. To identify how trees affected these soil properties, we evaluated correlations among species-level characteristics (e.g. nutrient concentrations in leaf litter, wood, and roots), stand-level properties (e.g. nutrient fluxes through leaf litterfall, nutrient pools in stemwood), and components of soil C, N, and cation cycles. Total extractable acidity (aciditytot) was correlated positively with mineral soil C stocks (R 2 = 0.72, P < 0.001), such that a nearly two-fold increase in aciditytot was associated with a more than two-fold increase of organic C. We attribute this correlation to effects of tree species on soil acidification and subsequent mineral weathering reactions, which make hydrolyzing cations available for stabilization of soil organic matter. The effects of tree species on soil acidity were better understood by measuring multiple components of soil acidity, including pH, the abundance of hydrolyzing cations in soil solutions and on cation exchange sites, and aciditytot. Soil pH and aciditytot were correlated with proton-producing components of the soil N cycle (e.g. nitrification), which were positively correlated with species-level variability in fine root N concentrations. Soluble components of soil acidity, such as aluminum in saturated paste extracts, were more strongly related to plant traits associated with calcium cycling, including leaf and root calcium concentrations. Our results suggest conceptual models of plant impacts on soil biogeochemistry should be revised to account for underappreciated plant traits and biogeochemical processes. 相似文献
95.
Piechota M Banach M Irzmański R Misztal M Rysz J Barylski M Piechota-Urbańska M Kowalski J Pawlicki L 《Cellular & molecular biology letters》2007,12(2):162-175
The aim of this study was to find the relationship between N-terminal brain natriuretic propeptide (NT-proBNP), procalcitonin
(PCT) and C-reactive protein (CRP) plasma concentrations in septic patients. This was a prospective study, performed at Medical
University Hospital No. 5 in łódź. Twenty patients with sepsis and severe sepsis were included in the study. N-terminal brain
natriuretic propeptide, procalcitonin and C-reactive protein concentrations, and survival were evaluated. In the whole studied
group (128 measurements), the mean NT-proBNP, procalcitonin and C-reactive protein concentrations were, respectively: 140.80±84.65
pg/ml, 22.32±97.41 ng/ml, 128.51±79.05 mg/l. The correlations for the NT-proBNP level and procalcitonin and C-reactive protein
levels were 0.3273 (p<0.001) and 0.4134 (p<0.001), respectively. NT-proBNP levels correlate with PCT and CRP levels in septic
patients. In the survivor subgroup, the mean NT-proBNP plasma concentrations were significantly lower than in the non-survivor
subgroup. 相似文献
96.
Shi L Bielawski J Mu J Dong H Teng C Zhang J Yang X Tomishige N Hanada K Hannun YA Zuo J 《Cell research》2007,17(12):1030-1040
Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B1 r_esistant11-1 (/br11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B l (FB0, a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain base 1 (LCB 1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr11-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FBl. By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine- 1-phosphate in a dosedependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues. 相似文献
97.
Hrebícek M Mrázová L Seyrantepe V Durand S Roslin NM Nosková L Hartmannová H Ivánek R Cízkova A Poupetová H Sikora J Urinovská J Stranecký V Zeman J Lepage P Roquis D Verner A Ausseil J Beesley CE Maire I Poorthuis BJ van de Kamp J van Diggelen OP Wevers RA Hudson TJ Fujiwara TM Majewski J Morgan K Kmoch S Pshezhetsky AV 《American journal of human genetics》2006,79(5):807-819
Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane. 相似文献
98.
In recent years, the bank voleMyodes glareolus (Schreber, 1780) has emerged as a model system for parasitological, behavioural and ecological studies and seems ideally
suited to address questions concerning the importance of MHC variation at individual and population levels. Here, we provide
the first extensive survey of sequence variation in the MHC class II DRB genes in this species. Among 34 analysed voles we
found 15 unique sequences, representing most likely two loci, at least one of them expressed. Despite very high overall sequence
divergence, particularly in the Antigen Binding Sites (ABS), we detected signatures of positive selection that has been acting
on DRB in the bank vole. Phylogenetic analysis demonstrated that the bank vole DRB alleles do not form a monophyletic group
but are intermingled with other rodent alleles that is consistent with long-term persistence of ancient allelic lineages maintained
through balancing selection. Our sequence data will forward the design of efficient genotyping methods, which will permit
testing hypotheses pertaining to the ecological causes and consequences of MHC variation in the bank vole. 相似文献
99.
Darzynkiewicz ZM Bojarska E Stepinski J Jemielity J Jankowska-Anyszka M Davis RE Darzynkiewicz E 《Nucleosides, nucleotides & nucleic acids》2007,26(10-12):1349-1352
Eukaryotic cells utilize scavenger decapping enzymes to degrade cap structure following 3'-5' mRNA decay. Human DcpS recently has been described as a highly specific hydrolase (a member of the HIT family) that catalyses the cleavage of m(7)GpppG and short capped oligoribonucleotides. We have demonstrated here that cap-1 (m(7)GpppGm) is a preferred substrate among several investigated dinucleotide cap analogues m(7)Gp(n)N (n = 3-5, N is a purine or pyrimidine base) and m(7)GMP is always one of the reaction product. Cap analogues containing pyrimidine base instead of guanine or diphosphate chain are resistant to hydrolysis catalyzed by human scavenger. Contrary to the other enzymes of HIT family, hDcpS activity is not stimulated by Mg(2+). 相似文献
100.
Ewa Kamler Rafa? Kamiński Jacek Wolnicki Justyna Sikorska Jakub Wa?owski 《Reviews in Fish Biology and Fisheries》2012,22(3):767-777
Six feeding groups of 60 early juveniles (0.6?g) of a rheophilic cyprinid barbel Barbus barbus were reared in triplicate in 18?glass aquaria. Fish fed a commercial formulated dry diet Aller Futura were compared with those on natural food??commercially available frozen Chironomidae larvae at 17, 21 and 25°C. Daily food rations were adjusted according to fish biomass, differences in hydration between the two diets, and rearing temperature. No mortality occurred during experiment. Temperature, and especially diet, both affected whole body proximate and mineral composition of B. barbus juveniles. Under conditions of this experiment the formulated diet Aller Futura seems to satisfy the demands for calcium and magnesium, while an elevated lipid content in this diet was found and marginally deficient phosphorus content can be concluded. These resulted in a depressed content of phosphorus and total minerals in the body, and in elevated lipid content and condition coefficient in the Futura-fed fish, but no visible body deformities occurred. Advantages and limitations of the condition coefficient K were discussed. This is a non-destructive, express indicator that can be used as a supplementary tool for estimation of changes in body proximate composition in fish juveniles of similar size within a population. 相似文献