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31.
Helena Santos-Rosa Gonzalo Millán-Zambrano Namshik Han Tommaso Leonardi Marie Klimontova Simona Nasiscionyte Luca Pandolfini Kostantinos Tzelepis Till Bartke Tony Kouzarides 《Molecular cell》2021,81(13):2793-2807.e8
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32.
M L Gonzales A Basu G H de Haas R Dijkman M G van Oort A A Okolo R H Glew 《Archives of biochemistry and biophysics》1988,262(1):345-353
The study of the acidic lipid requirement of human spleen glucocerebrosidase was extended to include two new series of acidic lipids, namely, monoacylglycol sulfates and diacylglycerol sulfates. Lysosomal glucocerebrosidase was extracted with sodium cholate and 1-butanol to render its beta-glucosidase activity dependent upon exogenous lipids. Maximum reactivation of control glucocerebrosidase was obtained with nonanoylglycol sulfate (NGS) and diheptanoylglycerol sulfate (DHGS). However, the effects of these lipids were markedly dependent on the nature of buffer used in the assay medium; specifically, 0.2 M sodium citrate-phosphate (pH 5.5) was much more effective than 0.2 M sodium acetate (pH 5.5) in permitting these lipids to reactivate glucocerebrosidase. In contrast, the marked activation of glucocerebrosidase by phosphatidylserine and galactocerebroside 3-sulfate (sulfatide) that was achievable in the sodium acetate buffer was totally inhibited by citrate or phosphate ions. The effects of NGS and DHGS on the kinetic parameters of control glucocerebrosidase were to lower the Km for the substrate, 4-methylumbelliferyl-beta-D-glucoside from 5.5 mM to approximately 2 mM (in sodium citrate-phosphate buffer) and markedly increase the Vmax. Furthermore, with DHGS, significant activation was achieved at concentrations below the lipid's critical micellar concentration. None of the monoacylglycol- or diacylglycerol sulfates were capable of stimulating mutant glucocerebrosidases from either type 1 (Ashkenazi-Jewish) or type 2 Gaucher's disease patients. Like control glucocerebrosidase, the type 1 glucocerebrosidase was unresponsive to phosphatidylserine and sulfatide when the beta-glucosidase assay was conducted in 0.2 M sodium citrate-phosphate buffer. Based on the differential action of these lipid activators in the two buffers and their effects on the mutant enzymes, we propose that, with regard to the lipid requirement of glucocerebrosidase, there are two classes of acidic lipids--one comprised of phosphatidylserine and sulfatide and the other comprised of the likes of NGS, DHGS, or sodium taurodeoxycholate. It appears that control glucocerebrosidase and the mutant enzyme of the patient with type 1 Gaucher's disease is reconstitutable with the first class of lipids whereas the glucocerebrosidase of the type 2 patient is not. The observations in this report are interpreted in terms of a model which postulates that normal glucocerebrosidase possesses at least two distinct lipid binding domains. 相似文献
33.
Hayley L. Crowell Katherine C. King James M. Whelan Mallory V. Harmel Gennesee Garcia Sebastian G. Gonzales Paul H. Maier Heather Neldner Thomas Nhu John T. Nolan Emily N. Taylor 《Ecology and evolution》2021,11(12):8170
- Most studies on how rising temperatures will impact terrestrial ectotherms have focused on single populations or multiple sympatric species. Addressing the thermal and energetic implications of climatic variation on multiple allopatric populations of a species will help us better understand how a species may be impacted by altered climates.
- We used eight years of thermal and behavioral data collected from four populations of Pacific rattlesnakes (Crotalus oreganus) living in climatically distinct habitat types (inland and coastal) to determine the field‐active and laboratory‐preferred body temperatures, thermoregulatory metrics, and maintenance energetic requirements of snakes from each population.
- Physical models showed that thermal quality was best at coastal sites, but inland snakes thermoregulated more accurately despite being in more thermally constrained environments. Projected increases of 1 and 2°C in ambient temperature result in an increase in overall thermal quality at both coastal and inland sites.
- Population differences in modeled standard metabolic rate estimates were driven by body size and not field‐active body temperature, with inland snakes requiring 1.6× more food annually than coastal snakes.
- All snakes thermoregulated with high accuracy, suggesting that small increases in ambient temperature are unlikely to impact the maintenance energetic requirements of individual snakes and that some species of large‐bodied reptiles may be robust to modest thermal perturbations under conservative climate change predictions.
34.
Borja Mateos Ganeko Bernardo-Seisdedos Valentin Dietrich Nicanor Zalba Gabriel Ortega Francesca Peccati Gonzalo Jiménez-Osés Robert Konrat Martin Tollinger Oscar Millet 《Biophysical journal》2021,120(10):2067-2077
Protein oligomerization processes are widespread and of crucial importance to understand degenerative diseases and healthy regulatory pathways. One particular case is the homo-oligomerization of folded domains involving domain swapping, often found as a part of the protein homeostasis in the crowded cytosol, composed of a complex mixture of cosolutes. Here, we have investigated the effect of a plethora of cosolutes of very diverse nature on the kinetics of a protein dimerization by domain swapping. In the absence of cosolutes, our system exhibits slow interconversion rates, with the reaction reaching the equilibrium within the average protein homeostasis timescale (24–48 h). In the presence of crowders, though, the oligomerization reaction in the same time frame will, depending on the protein's initial oligomeric state, either reach a pure equilibrium state or get kinetically trapped into an apparent equilibrium. Specifically, when the reaction is initiated from a large excess of dimer, it becomes unsensitive to the effect of cosolutes and reaches the same equilibrium populations as in the absence of cosolute. Conversely, when the reaction starts from a large excess of monomer, the reaction during the homeostatic timescale occurs under kinetic control, and it is exquisitely sensitive to the presence and nature of the cosolute. In this scenario (the most habitual case in intracellular oligomerization processes), the effect of cosolutes on the intermediate conformation and diffusion-mediated encounters will dictate how the cellular milieu affects the domain-swapping reaction. 相似文献
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39.
Hianara A. Bustamante Andrés Rivera-Dictter Viviana A. Cavieres Vanessa C. Mu?oz Alexis González Yimo Lin Gonzalo A. Mardones Patricia V. Burgos 《PloS one》2013,8(12)
Alzheimer’s disease (AD) is characterized by the buildup of amyloid-β peptides (Aβ) aggregates derived from proteolytic processing of the β-amyloid precursor protein (APP). Amyloidogenic cleavage of APP by β-secretase/BACE1 generates the C-terminal fragment C99/CTFβ that can be subsequently cleaved by γ-secretase to produce Aβ. Growing evidence indicates that high levels of C99/CTFβ are determinant for AD. Although it has been postulated that γ-secretase-independent pathways must control C99/CTFβ levels, the contribution of organelles with degradative functions, such as the endoplasmic reticulum (ER) or lysosomes, is unclear. In this report, we investigated the turnover and amyloidogenic processing of C99/CTFβ in human H4 neuroglioma cells, and found that C99/CTFβ is localized at the Golgi apparatus in contrast to APP, which is mostly found in endosomes. Conditions that localized C99/CTFβ to the ER resulted in its degradation in a proteasome-dependent manner that first required polyubiquitination, consistent with an active role of the ER associated degradation (ERAD) in this process. Furthermore, when proteasomal activity was inhibited C99/CTFβ was degraded in a chloroquine (CQ)-sensitive compartment, implicating lysosomes as alternative sites for its degradation. Our results highlight a crosstalk between degradation pathways within the ER and lysosomes to avoid protein accumulation and toxicity. 相似文献
40.
Miguel F. Gonzales Teresa Brooks Stefan U. Pukatzki Daniele Provenzano 《Journal of visualized experiments : JoVE》2013,(80)
Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community. 相似文献