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61.
The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.  相似文献   
62.
Insulin receptor substrate (IRS) 2 as intermediate docking platform transduces the insulin/IGF-1 (insulin like growth factor 1) signal to intracellular effector molecules that regulate glucose homeostasis, β-cell growth, and survival. Previously, IRS2 has been identified as a 14-3-3 interaction protein. 14-3-3 proteins can bind their target proteins via phosphorylated serine/threonine residues located within distinct motifs. In this study the binding of 14-3-3 to IRS2 upon stimulation with forskolin or the cAMP analog 8-(4-chlorophenylthio)-cAMP was demonstrated in HEK293 cells. Binding was reduced with PKA inhibitors H89 or Rp-8-Br-cAMPS. Phosphorylation of IRS2 on PKA consensus motifs was induced by forskolin and the PKA activator N6-Phe-cAMP and prevented by both PKA inhibitors. The amino acid region after position 952 on IRS2 was identified as the 14-3-3 binding region by GST-14-3-3 pulldown assays. Mass spectrometric analysis revealed serine 1137 and serine 1138 as cAMP-dependent, potential PKA phosphorylation sites. Mutation of serine 1137/1138 to alanine strongly reduced the cAMP-dependent 14-3-3 binding. Application of cycloheximide revealed that forskolin enhanced IRS2 protein stability in HEK293 cells stably expressing IRS2 as well as in primary hepatocytes. Stimulation with forskolin did not increase protein stability either in the presence of a 14-3-3 antagonist or in the double 1137/1138 alanine mutant. Thus the reduced IRS2 protein degradation was dependent on the interaction with 14-3-3 proteins and the presence of serine 1137/1138. We present serine 1137/1138 as novel cAMP-dependent phosphorylation sites on IRS2 and show their importance in 14-3-3 binding and IRS2 protein stability.  相似文献   
63.
Abstract: The molecular origin of protein stability has been the subject of active research for more than a generation (R. Jaenicke (1991) Eur. J. Biochem. 202, 715–728). Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential. In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima , it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds ( ΔΔG stab≈ 50 kJ/mol). As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology. In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents.  相似文献   
64.
The capacity of the -Proteobacterium Pseudomonas sp. strain P51, which degrades chlorinated benzenes, to metabolize 1,2,4-trichlorobenzene (TCB) under environmental conditions was tested by its release into two experimental systems. The first system consisted of laboratory scale microcosms which were operated with and without the addition of TCB and which were inoculated with sludge from a wastewater treatment plant. The second system consisted of a non sterile, water saturated soil column. We determined survival of strain P51 after its introduction and its ability to degrade TCB. The population dynamics was followed by selective plating and applying the polymerase chain reaction (PCR) to detect strain P51 and the chlorobenzene ( tcb) genes on catabolic plasmid pP51. The results showed a completely different behaviour of strain P51 in the two habitats under the applied conditions. In the soil column the P51 bacteria inoculated the entire area and their population reached 2 × 106 cells/g soil. The population remained active since TCB was degraded to concentrations below the detection limit of 30 g/l. In the sludge microcosms, the number of strain P51 cells immediately decreased from 4 × 107 cells/ml to 105 cells/ml over a period of 2 days after inoculation, and then the strain disappeared to levels below our detection limit (103–104 cells/ml). In the reactor without TCB the population of P51 maintained a stable value of 105 cells/ml during 8 days but then also decreased to levels below the detection limit. In addition, no significant TCB degradation was found in the sludge reactors. The influence of presence of TCB on maintenance of strain P51 in the two habitats is discussed. This work demonstrates the possibility to successfully apply preselected strains to degrade otherwise poorly degradable substances in complex mixed microbial communities. However, survival and activity may depend strongly on the type of system into which the strain is introduced.  相似文献   
65.
The unknown status of inland fish stocks hinders their sustainable management. Therefore, increasing stock status information is important for sustainable inland fisheries. Fisheries reference points were estimated for five exploited fish species (11 stocks) in the Lake Edward system, East Africa, which is one of the most productive inland water systems. The aim was to ascertain the status of the fisheries and establish reference points for effective management. The reference points were based on four linked stock assessment approaches for data-limited fisheries. Estimates showed poor stock status with the stocks defined as either collapsed, recruitment impaired or overfished. However, higher catches could be obtained under sustainable management. Estimates of maximum sustainable yield (MSY) and supporting biomass (Bmsy) are provided for 10 of the stocks as targets for rebuilding plans. The immediate target of management should be rebuilding biomass to Bmsy. Applicable measures include shifting length at first capture to the length that maximizes catch without endangering size structure and biomass, and livelihood diversification out of fisheries.  相似文献   
66.
Apple replant disease (ARD) is common to all major apple-growing regions in the world. It occurs when new apple trees are replanted on sites where previously the same or closely related crop species were grown. Biotic (fungi, bacteria and nematodes) and abiotic soil factors (poor soil structure, nutrition) contribute to the development and severity of ARD. However, the aetiology of ARD and effects on higher trophic levels are still unknown. In that sense, Collembola might play an important role, since they are one of the dominant mesofauna groups in many soils. They act as decomposer, fungivores and predators, representing different trophic levels in soil food webs. Therefore, any effect of ARD on the occurrence of Collembola could have ecological impacts on the soil quality and health. Here, we examined the colonization behaviour of two Collembolan species, Folsomia candida and Sinella curviseta, in choice tests and population growth tests using Apple Replant Diseased soil (ARD) and non-ARD soil samples from different field sites and standardized laboratory bioassays. Additionally, Collembola behaviour was quantified by continuous video observations to investigate short-term behavioural changes. Results showed that both Collembolan species significantly preferred colonization of the non-ARD soils compared with ARD soils, independent of the origin of the soil samples or specific disinfection treatments. Moreover, the detailed video analysis of the foraging behaviour indicates rapid colonization of soil samples and low dispersal rates. Most likely, volatile compounds and to a lesser extent feeding stimulants play a vital role for the colonization process for both Collembolan species. Finally, results showed negative effects of ARD on population growth of both Collembolan species already after an 8-week period, implying strong nutritional deficiencies in ARD affected soils. The hypothesis that ARD causing microorganisms directly affected orientation, colonization and population development of Collembola is discussed.  相似文献   
67.
Abstract: Cyclic GMP (cGMP) formation in rat pinealocytes is regulated through a synergistic dual receptor mechanism involving β-and α1-adrenergic receptors. The effects of N -monomethyl- l -arginine (NMMA), which inhibits nitric oxide (NO) synthase and NO-mediated activation of cytosolic guanylate cyclase, and methylene blue (MB), which inhibits cytosolic guanylate cyclase, were investigated in an attempt to understand the role of NO in adrenergic cGMP formation. Both NMMA and MB inhibited β-adrenergic stimulation of cGMP formation as well as α1-adrenergic potentiation of β-adrenergic stimulation of cGMP formation, whereas they had no effect in unstimulated pinealocytes. The inhibitory action of NMMA was antagonized by addition of l -arginine. On the basis of these findings it can be concluded that the adrenergic stimulation of cGMP formation involves NO synthesis followed by activation of cytosolic guanylate cyclase.  相似文献   
68.
Sun Y  Lim Y  Li F  Liu S  Lu JJ  Haberberger R  Zhong JH  Zhou XF 《PloS one》2012,7(4):e35883

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.  相似文献   
69.
70.
Lipoic acid was recently demonstrated to improve endothelial dysfunction or retinopathy not only in rats but also in diabetic patients. We tested the hypothesis that R-(+)-alpha-lipoic acid (LA) directly affects human endothelial cell (EC) function (e.g., apoptosis, proliferation, and protein expression), independent of the cells' vascular origin. Macrovascular EC (macEC), isolated from umbilical (HUVEC) and adult saphenous veins and from aortae, as well as microvascular EC (micEC) from retinae, skin, and uterus, were exposed to LA (1 mumol/l-1 mmol/l) with/without different stimuli (high glucose, TNF-alpha, VEGF, wortmannin, LY-294002). Apoptosis, proliferation, cell cycle distribution, and protein expression were determined by DNA fragmentation assays, [(3)H]thymidine incorporation, FACS, and Western blot analyses, respectively. In macro- and microvascular EC, LA (1 mmol/l) reduced (P < 0.05) basal (macEC, -36 +/- 4%; micEC, -46 +/- 6%) and stimulus-induced (TNF-alpha: macEC, -75 +/- 11%; micEC, -68 +/- 13%) apoptosis. In HUVEC, inhibition of apoptosis by LA (500 mumol/l) was paralleled by reduction of NF-kappaB. LA's antiapoptotic activity was reduced by PI 3-kinase inhibitors (wortmannin, LY-294002), being in line with LA-induced Akt phosphorylation (Ser(437), +159 +/- 43%; Thr(308), +98 +/- 25%; P < 0.01). LA (500 mumol/l) inhibited (P < 0.001) proliferation of macEC (-29 +/- 3%) and micEC (-29 +/- 3%) by arresting the cells at the G(1)/S transition due to an increased ratio of cyclin E/p27(Kip) (4.2-fold), upregulation of p21(WAF-1/Cip1) (+104 +/- 21%), and reduction of cyclin A (-32 +/- 11%), of hyperphosphorylated retinoblastoma protein (macEC: -51 +/- 7%; micEC: -50 +/- 15%), and of E2F-1 (macEC: -48 +/- 3%; micEC: -31 +/- 10%). LA's ability to inhibit apoptosis and proliferation of ECs could beneficially affect endothelial dysfunction, which precedes manifestation of late diabetic vascular complications.  相似文献   
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