蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

Failure and moral distinction in a Ukrainian marketplace of ideas

This essay examines the generative effects of claiming moral failure within a Ukrainian liberal movement for media reform in post-Maidan, pre-invasion Ukraine. The reformers wished to reorganize news reporting around the ideals of autonomy, balanced objectivity, impartiality, and corrigibility, which they believed underpinned Western media. They decried most Ukrainian media as failing such standards, highlighting the individual moral failure of journalists bankrolled by oligarchs in return for favourable media representation. In turn, those from whom the reformers tried to distinguish themselves morally mocked them as ‘grant-eaters’ for their dependence on Western democracy promotion grants. This tussle pitted material success against yearnings for moral and professional probity. Developing Selka's idea of moral distinction, I argue that while the reformers’ pursuit of virtuous difference was sincere, their structural vulnerability vis-à-vis the mainstream media also made morality more salient as the basis for agonistic differentiation.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Reconstructing Native American Migrations from Whole-Genome and Whole-Exome Data

There is great scientific and popular interest in understanding the genetic history of populations in the Americas. We wish to understand when different regions of the continent were inhabited, where settlers came from, and how current inhabitants relate genetically to earlier populations. Recent studies unraveled parts of the genetic history of the continent using genotyping arrays and uniparental markers. The 1000 Genomes Project provides a unique opportunity for improving our understanding of population genetic history by providing over a hundred sequenced low coverage genomes and exomes from Colombian (CLM), Mexican-American (MXL), and Puerto Rican (PUR) populations. Here, we explore the genomic contributions of African, European, and especially Native American ancestry to these populations. Estimated Native American ancestry is in MXL, in CLM, and in PUR. Native American ancestry in PUR is most closely related to populations surrounding the Orinoco River basin, confirming the Southern America ancestry of the Taíno people of the Caribbean. We present new methods to estimate the allele frequencies in the Native American fraction of the populations, and model their distribution using a demographic model for three ancestral Native American populations. These ancestral populations likely split in close succession: the most likely scenario, based on a peopling of the Americas thousand years ago (kya), supports that the MXL Ancestors split kya, with a subsequent split of the ancestors to CLM and PUR kya. The model also features effective populations of in Mexico, in Colombia, and in Puerto Rico. Modeling Identity-by-descent (IBD) and ancestry tract length, we show that post-contact populations also differ markedly in their effective sizes and migration patterns, with Puerto Rico showing the smallest effective size and the earlier migration from Europe. Finally, we compare IBD and ancestry assignments to find evidence for relatedness among European founders to the three populations.     

Application of docking methodologies to modeled proteins

Protein docking is essential for structural characterization of protein interactions. Besides providing the structure of protein complexes, modeling of proteins and their complexes is important for understanding the fundamental principles and specific aspects of protein interactions. The accuracy of protein modeling, in general, is still less than that of the experimental approaches. Thus, it is important to investigate the applicability of docking techniques to modeled proteins. We present new comprehensive benchmark sets of protein models for the development and validation of protein docking, as well as a systematic assessment of free and template-based docking techniques on these sets. As opposed to previous studies, the benchmark sets reflect the real case modeling/docking scenario where the accuracy of the models is assessed by the modeling procedure, without reference to the native structure (which would be unknown in practical applications). We also expanded the analysis to include docking of protein pairs where proteins have different structural accuracy. The results show that, in general, the template-based docking is less sensitive to the structural inaccuracies of the models than the free docking. The near-native docking poses generated by the template-based approach, typically, also have higher ranks than those produces by the free docking (although the free docking is indispensable in modeling the multiplicity of protein interactions in a crowded cellular environment). The results show that docking techniques are applicable to protein models in a broad range of modeling accuracy. The study provides clear guidelines for practical applications of docking to protein models.     

Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors

The development of high‐performance photobioreactors equipped with automatic systems for non‐invasive real‐time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light‐induced fluorescence rise and the dark relaxation of the flash‐induced fluorescence yield (Qa^? ? re‐oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas‐tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long‐term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress‐sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real‐time monitoring of algal photosynthesis in photobioreactors.     

Role of the nitric oxide/cyclic GMP pathway and extracellular environment in the nitric oxide donor-induced increase in dopamine secretion from PC12 cells: a microdialysis in vitro study

In vitro microdialysis was used to investigate the mechanism of nitric oxide (NO) donor-induced changes in dopamine (DA) secretion from PC12 cells. Infusion of the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 1.0 mm) induced a long-lasting increase in DA and 3-methoxytyramine (3-MT) dialysate concentrations. SNAP-induced increases were inhibited either by pre-infusion of the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4] oxadiazolo[4,3]quinoxalin-1-one (ODQ, 0.1 mm) or by Ca2+ omission. Ca2+ re-introduction restored SNAP effects. SNAP-induced increases in DA + 3-MT were unaffected by co-infusion of the l-type Ca2+ channel inhibitor nifedipine. The NO-donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3, 1.0 mm) induced a short-lasting decrease in dialysate DA + 3-MT. Ascorbic acid (0.2 mm) co-infusion allowed NOR-3 to increase dialysate DA + 3-MT. ODQ pre-infusion inhibited NOR-3 + ascorbic acid-induced DA + 3-MT increases. Infusion of high K+ (75 mm) induced a 2.5-fold increase in dialysate DA + 3-MT. The increase was abolished by NOR-3 co-infusion. Conversely, co-infusion of ascorbic acid (0.2 mm) with NOR-3 + high K+ restored high K+ effects. Co-infusion of nifedipine inhibited high K+-induced DA + 3-MT increases. These results suggest that activation of the NO/sGC/cyclic GMP pathway may be the underlying mechanism of extracellular Ca2+-dependent effects of exogenous NO on DA secretion from PC12 cells. Extracellular Ca2+ entry may occur through nifedipine-insensitive channels. NO effects and DA concentrations in dialysates largely depend on both the timing of NO generation and the extracellular environment in which NO is generated.     

Subcellular localization and regulation of coenzyme A synthase

CoA synthase mediates the last two steps in the sequence of enzymatic reactions, leading to CoA biosynthesis. We have recently identified cDNA for CoA synthase and demonstrated that it encodes a bifunctional enzyme possessing 4'-phosphopantetheine adenylyltransferase and dephospho-CoA kinase activities. Molecular cloning of CoA synthase provided us with necessary tools to study subcellular localization and the regulation of this bifunctional enzyme. Transient expression studies and confocal microscopy allowed us to demonstrate that full-length CoA synthase is associated with the mitochondria, whereas the removal of the N-terminal region relocates the enzyme to the cytosol. In addition, we showed that the N-terminal sequence of CoA synthase (amino acids 1-29) exhibits a hydrophobic profile and targets green fluorescent protein exclusively to mitochondria. Further analysis, involving subcellular fractionation and limited proteolysis, indicated that CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.     

The Role of auxin,pH, and stress in the activation of embryogenic cell division in leaf protoplast-derived cells of alfalfa

Culturing leaf protoplast-derived cells of the embryogenic alfalfa (Medicago sativa subsp. varia A2) genotype in the presence of low (1 microM) or high (10 microM) 2, 4-dichlorophenoxyacetic acid (2,4-D) concentrations results in different cell types. Cells exposed to high 2,4-D concentration remain small with dense cytoplasm and can develop into proembryogenic cell clusters, whereas protoplasts cultured at low auxin concentration elongate and subsequently die or form undifferentiated cell colonies. Fe stress applied at nonlethal concentrations (1 mM) in the presence of 1 microM 2,4-D also resulted in the development of the embryogenic cell type. Although cytoplasmic alkalinization was detected during cell activation of both types, embryogenic cells could be characterized by earlier cell division, a more alkalic vacuolar pH, and nonfunctional chloroplasts as compared with the elongated, nonembryogenic cells. Buffering of the 10 microM 2,4-D-containing culture medium by 10 mM 2-(N-morpholino)ethanesulfonic acid delayed cell division and resulted in nonembryogenic cell-type formation. The level of endogenous indoleacetic acid (IAA) increased transiently in all protoplast cultures during the first 4 to 5 d, but an earlier peak of IAA accumulation correlated with the earlier activation of the division cycle in embryogenic-type cells. However, this IAA peak could also be delayed by buffering of the medium pH by 2-(N-morpholino)ethanesulfonic acid. Based on the above data, we propose the involvement of stress responses, endogenous auxin synthesis, and the establishment of cellular pH gradients in the formation of the embryogenic cell type.     

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.