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101.
Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion. 相似文献
102.
Phylogeography of Y-chromosome haplogroup I reveals distinct domains of prehistoric gene flow in europe 总被引:14,自引:2,他引:12 下载免费PDF全文
Rootsi S Magri C Kivisild T Benuzzi G Help H Bermisheva M Kutuev I Barać L Pericić M Balanovsky O Pshenichnov A Dion D Grobei M Zhivotovsky LA Battaglia V Achilli A Al-Zahery N Parik J King R Cinnioğlu C Khusnutdinova E Rudan P Balanovska E Scheffrahn W Simonescu M Brehm A Goncalves R Rosa A Moisan JP Chaventre A Ferak V Füredi S Oefner PJ Shen P Beckman L Mikerezi I Terzić R Primorac D Cambon-Thomsen A Krumina A Torroni A Underhill PA Santachiara-Benerecetti AS Villems R Semino O 《American journal of human genetics》2004,75(1):128-137
To investigate which aspects of contemporary human Y-chromosome variation in Europe are characteristic of primary colonization, late-glacial expansions from refuge areas, Neolithic dispersals, or more recent events of gene flow, we have analyzed, in detail, haplogroup I (Hg I), the only major clade of the Y phylogeny that is widespread over Europe but virtually absent elsewhere. The analysis of 1,104 Hg I Y chromosomes, which were identified in the survey of 7,574 males from 60 population samples, revealed several subclades with distinct geographic distributions. Subclade I1a accounts for most of Hg I in Scandinavia, with a rapidly decreasing frequency toward both the East European Plain and the Atlantic fringe, but microsatellite diversity reveals that France could be the source region of the early spread of both I1a and the less common I1c. Also, I1b*, which extends from the eastern Adriatic to eastern Europe and declines noticeably toward the southern Balkans and abruptly toward the periphery of northern Italy, probably diffused after the Last Glacial Maximum from a homeland in eastern Europe or the Balkans. In contrast, I1b2 most likely arose in southern France/Iberia. Similarly to the other subclades, it underwent a postglacial expansion and marked the human colonization of Sardinia ~9,000 years ago. 相似文献
103.
Garcin ED Bruns CM Lloyd SJ Hosfield DJ Tiso M Gachhui R Stuehr DJ Tainer JA Getzoff ED 《The Journal of biological chemistry》2004,279(36):37918-37927
Three nitric-oxide synthase (NOS) isozymes play crucial, but distinct, roles in neurotransmission, vascular homeostasis, and host defense, by catalyzing Ca(2+)/calmodulin-triggered NO synthesis. Here, we address current questions regarding NOS activity and regulation by combining mutagenesis and biochemistry with crystal structure determination of a fully assembled, electron-supplying, neuronal NOS reductase dimer. By integrating these results, we structurally elucidate the unique mechanisms for isozyme-specific regulation of electron transfer in NOS. Our discovery of the autoinhibitory helix, its placement between domains, and striking similarities with canonical calmodulin-binding motifs, support new mechanisms for NOS inhibition. NADPH, isozyme-specific residue Arg(1400), and the C-terminal tail synergistically repress NOS activity by locking the FMN binding domain in an electron-accepting position. Our analyses suggest that calmodulin binding or C-terminal tail phosphorylation frees a large scale swinging motion of the entire FMN domain to deliver electrons to the catalytic module in the holoenzyme. 相似文献
104.
105.
Food web structure is well known to vary widely among ecosystems. Recent research indicates that there can be a high degree
of spatial heterogeneity within ecosystems as well. Xochimilco is a small heterogeneous freshwater system that has been transformed
into a network of canals, small lakes, and wetlands. Located within Mexico City, this ecosystem has been intensively managed
and highly impacted for more than 50 years. This system receives urban and agricultural runoff, with resulting impacts on
water quality. The aquatic community is dominated by exotics such as carp (Cyprinus carpio) and tilapia (Oreocrhomis niloticus), though the system still supports endemic species such as the aquatic salamander, axolotl (Ambystoma mexicanum), and crayfish (Cambarellus montezumae), which are both endangered. In this study, we used carbon and nitrogen stable isotopes for the whole food web and gut content
analysis from the exotic fishes to describe food web structure in different canals within Xochimilco. There were significant
isotopic differences among canals. These differences may result from isotopic baseline differences as well as differences
in actual food web structure: both are related to local spatial variation in water quality driven by nutrient inputs and exotic
fishes. Within-ecosystem variability is likely to be seen in other perturbed shallow systems as well, and should be explicitly
considered in future food web studies. 相似文献
106.
107.
In this study we investigated the dynamic behavior of the chimeric cell-penetrating peptide transportan in membrane-like environments using NMR. Backbone amide 15N spin relaxation was used to investigate the dynamics in two bicelles: neutral DMPC bicelles and partly negatively charged DMPG-containing bicelles. The structure of the peptide as judged from CD and chemical shifts is similar in the two cases. Both the overall motion as well as the local dynamics is, however, different in the two types of bicelles. The overall dynamics of the peptide is significantly slower in the partly negatively charged bicelle environment, as evidenced by longer global correlation times for all measured sites. The local motion, as judged from generalized order parameters, is for all sites in the peptide more restricted when bound to negatively charged bicelles than when bound to neutral bicelles (increase in S
2 is on average 0.11 ± 0.07). The slower dynamics of transportan in charged membrane model systems cause significant line broadening in the proton NMR spectrum, which in certain cases limits the observation of 1H signals for transportan when bound to the membrane. The effect of transportan on DMPC and DHPC motion in zwitterionic bicelles was also investigated, and the motion of both components in the bicelle was found to be affected.Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s10858-006-9008-y and is accessible for authorized users. 相似文献
108.
Some studies have demonstrated that a few biological systems are affected by weak, extremely low frequency (ELF) electromagnetic fields (EMFs), lower than 10 mT. However, to date there is scanty evidence of this effect on Protists in the literature. Due to their peculiarity as single-cell eukaryotic organisms, Protists respond directly to environmental stimuli, thus appearing as very suitable experimental systems. Recently, we showed the presence of propionylcholinesterase (PrChE) activity in single-cell amoebae of Dictyostelium discoideum. This enzyme activity was assumed to be involved in cell-cell and cell-environment interactions, as its inhibition affects cell aggregation and differentiation. In this work, we have exposed single-cell amoebae of D. discoideum to an ELF-EMF of about 200 microT, 50 Hz, for 3 h or 24 h at 21 degrees C. A delay in the early phase of the differentiation was observed in 3 h exposed cells, and a significant decrease in the fission rate appeared in 24 h exposed cells. The PrChE activity was significantly lower in 3 h exposed cells than in the controls, whereas 24 h exposed cells exhibited an increase in this enzyme activity. However, such effects appeared to be transient, as the fission rate and PrChE activity values returned to the respective control values after a 24 h stay under standard conditions. 相似文献
109.
Laugesen S Messinese E Hem S Pichereaux C Grat S Ranjeva R Rossignol M Bono JJ 《Phytochemistry》2006,67(20):2208-2214
The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants. 相似文献
110.
Quantitative trait loci for grain moisture at harvest and field grain drying rate in maize (Zea mays, L.) 总被引:1,自引:0,他引:1
Sala RG Andrade FH Camadro EL Cerono JC 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2006,112(3):462-471
Hybrids with low grain moisture (GM) at harvest are specially required in mid- to short-season environments. One of the most
important factors determining this trait is field grain drying rate (FDR). To produce hybrids with low GM at harvest, inbred
lines can be obtained through selection for either GM or FDR. Thus, a single-cross population (181 F
2:3-generation plants) of two divergent inbred lines was evaluated to locate QTL affecting GM at harvest and FDR as a starting
point for marker assisted selection (MAS). Moisture measurements were made with a hand-held moisture meter. Detection of QTL
was facilitated with interval mapping in one and two dimensions including an interaction term, and a genetic linkage map of
122 SSR loci covering 1,557.8 cM. The markers were arranged in ten linkage groups. QTL mapping was made for the mean trait
performance of the F
2:3 population across years. Ten QTL and an interaction were associated with GM. These QTL accounted for 54.8 and 65.2% of the
phenotypic and genotypic variation, respectively. Eight QTL and two interactions were associated with FDR accounting for 35.7
and 45.2% of the phenotypic and genotypic variation, respectively. Two regions were in common between traits. The interaction
between QTL for GM at harvest had practical implications for MAS. We conclude that MAS per se will not be an efficient method
for reducing GM at harvest and/or increasing FDR. A selection index including both molecular marker information and phenotypic
values, each appropriately weighted, would be the best selection strategy. 相似文献