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Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn2+ metal ions enhances inclusion formation. Furthermore, Zn2+ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.  相似文献   
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One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.  相似文献   
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The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.  相似文献   
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Summary Genetic effects for varietal value are defined at the level of the population of k-parent synthetic varieties. A simple expression for the total variance among synthetics arises directly from these definitions. A general expression for the covariance among related synthetics is given. Genetic effects are also defined in a completely general way so as to allow for any system of testing and used to derive an expression for the genetic advance in recurrent selection for varietal value. Covariances between relatives evaluated in the system of testing and in varietal combination are introduced, allowing a direct expression of the genetic advance in varietal development when parents are selected either individually or in groups. Some general implications for plant breeding are outlined.Dedicated to Professor F.W. Schnell on the occasion of his 65th birthday  相似文献   
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Sera from three chimpanzees infected with a primary lymphadenopathy-associated virus (LAV-1) or human T-lymphotropic virus type III (HTLV-IIIB) passage, from two chimpanzees infected with blood from the primary infected chimpanzees, and from one chimpanzee infected with blood from a secondary passage animal all bound the peptides 3B and 3B/RF, sharing the sequence IQRGPGR, with equally high titers. Pepscan analysis confirmed the amino acids Q, R, G, P, and G as irreplaceable in order to retain antigenicity.  相似文献   
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Summary Single photoreceptor cells in the compound eye of the housefly Musca domestica were selectively illuminated and subsequently compared electron-microscopically with the unilluminated photoreceptors in the immediate surroundings. The rhabdomeres of the illuminated cells remain largely unaffected, but the cells show an increase in the number of coated pits, various types of vesicles, and degradative organelles; some of the latter organelles are described for the first time in fly photoreceptors. Coated pits are found not only at the bases of the microvilli, but also in other parts of the plasma membrane. Degradative organelles, endoplasmic reticulum (ER) and mitochondria aggregate in the perinuclear region. The rough ER and smooth ER are more elaborate, the number of Golgi stacks, free ribosomes and polysomes is increased, and the shape and distribution of heterochromatin within the nuclei are altered. Illuminated photoreceptors also interdigitate extensively with their neighbouring secondary pigment cells. These structural changes in illuminated fly photoreceptor cells indicate an increase in membrane turnover and cellular metabolism. When applied to the eye, Lucifer Yellow spreads into the extracellular space and is taken up only by the illuminated photoreceptor cells. These cells show the same structural modifications as above. Horseradish peroxidase applied in the same way is observed in pinocytotic vesicles and degradative organelles of the illuminated cells. Hence, the light-induced uptake of extracellular compounds takes place in vivo at least partially as a result of an increase in pinocytosis.  相似文献   
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Summary The epidermal Merkel cells and their sensory innervation serve tactile sensation in vertebrates. In this study the fluorescent cationic mitochondrial dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), which has recently been used as a vital stain for motor and autonomic nerve terminals, was tested for its ability to stain Merkel cells and sensory fibers in the snout of the rat. Brightly-fluorescent structures resembling Merkel cells as well as nerve fibers and their terminations were evident in whole mounts of the vibrissal follicle. Unilateral denervation of the vibrissal follicles soon after birth resulted in a staining pattern remarkably similar to that obtained after labelling of the Merkel cells selectively with the fluorescent marker quinacrine, but all fiber staining was abolished. Likewise, in the separated epidermis of other skin regions, including the hairy and glabrous skin of the nose, the staining pattern revealed by 4-Di-2-ASP was indistinguishable from that obtained by quinacrine fluorescence. These results indicate that certain styryl pyridinium dyes may be used as vital stains for epidermal Merkel cells as well as cutaneous mechanosensory axons.  相似文献   
10.
In order to improve the generating and photochemical properties of coumarin laser dyes, the following active media were synthesized: inclusion complexes of -cyclodextrin (-CD) and 7-amino-4-methylcoumarins (COU1, COU102, COU120). Complex formation processes were studied, and the structure of the inclusion complexes was estimated using the method of MM2 molecular mechanics. The data obtained suggest the reasons underlying the complex structure effects on their spectral, luminescent and generating characteristics.  相似文献   
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