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. 相似文献
In this paper, a graphene-based tunable multi-band terahertz absorber is proposed and numerically investigated. The proposed absorber can achieve perfect absorption within both sharp and ultra-broadband absorption spectra. This wide range of absorption is gathered through a unique combination of periodically cross- and square-shaped dielectrics sandwiched between two graphene sheets; the latter enables it to offer more absorption in comparison with the traditional single-layer graphene structures. The aforementioned top layer is mounted on a gold plate separated by a Topas layer with zero volume loss. Furthermore, in our proposed approach, we investigated the possibility of changing the shapes and sizes of the dielectric layers instead of the geometry of the graphene layers to alleviate the edge effects and manufacturing complications. In numerical simulations, parameters, such as graphene Fermi energy and the dimensions of the proposed dielectric layout, have been optimally tuned to reach perfect absorption. We have verified that the performance of our dielectric layout called fishnet, with two widely investigated dielectric layouts in the literature (namely, cross-shaped and frame-and-square). Our results demonstrate two absorption bands with near-unity absorbance at frequencies of 1.6–2.3 and 4.2–4.9 THz, with absorption efficiency of 98% in 1.96 and 4.62 THz, respectively. Moreover, a broadband absorption in the 7.77–9.78 THz is observed with an absorption efficiency of 99.6% that was attained in 8.44–9.11 THz. Finally, the versatility provided by the tunability of three operation bands of the absorber makes it a great candidate for integration into terahertz optoelectronic devices.
Schwann cells (SCs), the supporting cells of the peripheral nerves, are indispensable for regenerating the peripheral and central nervous system. Copious preparation of these cells in a well-defined manner is to be a privileged position. SCs cultivation is overwhelmed by contaminating fibroblasts which are often outgrowing as the predominant cell type in an in vitro culture. This study introduces a technically simple and efficient procedure for SCs isolation and enrichment based on implementing recombinant and defined supplements. Collected adult rat sciatic nerves were cultured for 10 days as in vitro predegeneration. After dissociation and plating, the medium changed to knockout serum replacement supplemented DMDM/F12 medium containing various growth factors. The whole procedure took 3 weeks and SCs purity was then evaluated through implementing specific cytoplasmic and membranous markers. The viability of enriched SCs were evaluated by MTT assay. Within 10 days, over 99 % homogenous SCs were achieved and confirmed through immunofluorescence staining and flow-cytometry for P75NTR and S100 markers, respectively. MTT data revealed that the viability and metabolic activities of purified SCs were increased in expansion medium. This study provides a technically easy and efficient method with the benefits of not utilizing bovine serum or other animal products for SCs isolation and enrichment. 相似文献
Phylogenetic relationships were determined for 76 partial P-element
sequences from 14 species of the melanogaster species group within the
Drosophila subgenus Sophophora. These results are examined in the context
of the phylogeny of the species from which the sequences were isolated.
Sequences from the P-element family fall into distinct subfamilies, or
clades, which are often characteristic for particular species subgroups.
When examined locally among closely related species, the evolution of P
elements is characterized by vertical transmission, whereby the P-element
phylogeny traces the species phylogeny. On a broader scale, however, the
P-element phylogeny is not congruent with the species phylogeny. One
feature of P-element evolution in the melanogaster group is the presence of
more than one P-element subfamily, differing by as much as 36%, in the
genomes of some species. Thus, P elements from several individual species
are not monophyletic, and a likely explanation for the incongruence between
P-element and species phylogenies is provided by the comparison of
paralogous sequences. In certain instances, horizontal transfer seems to be
a valid alternative explanation for lack of congruence between species and
P-element phylogenies. The canonical P-element subfamily, which represents
the active, autonomous transposable element, is restricted to D.
melanogaster. Thus, its origin clearly lies outside of the melanogaster
species group, consistent with the earlier conclusion of recent horizontal
transfer.
相似文献
Cellular responses to death-promoting stimuli typically proceed through a differentiated multistage process, involving a lag
phase, extensive death, and potential adaptation. Deregulation of this chain of events is at the root of many diseases. Improper
adaptation is particularly important because it allows cell sub-populations to survive even in the continuous presence of
death conditions, which results, among others, in the eventual failure of many targeted anticancer therapies. 相似文献
Multiple congenital ocular anomalies (MCOA) syndrome is a hereditary congenital eye defect that was first described in Silver colored Rocky Mountain horses. The mutation causing this disease is located within a defined chromosomal interval, which also contains the gene and mutation that is associated with the Silver coat color (PMEL17, exon 11). Horses that are homozygous for the disease-causing allele have multiple defects (MCOA-phenotype), whilst the heterozygous horses predominantly have cysts of the iris, ciliary body or retina (Cyst-phenotype). It has been argued that these ocular defects are caused by a recent mutation that is restricted to horses that are related to the Rocky Mountain Horse breed. For that reason we have examined another horse breed, the Icelandic horse, which is historically quite divergent from Rocky Mountain horses.
Results
We examined 24 Icelandic horses and established that the MCOA syndrome is present in this breed. Four of these horses were categorised as having the MCOA-phenotype and were genotyped as being homozygous for the PMEL17 mutation. The most common clinical signs included megaloglobus, iris stromal hypoplasia, abnormal pectinate ligaments, iridociliary cysts occasionally extending into the peripheral retina and cataracts. The cysts and pectinate ligament abnormalities were observed in the temporal quadrant of the eyes. Fourteen horses were heterozygous for the PMEL17 mutation and were characterized as having the Cyst-phenotype with cysts and occasionally curvilinear streaks in the peripheral retina. Three additional horses were genotyped as PMEL17 heterozygotes, but in these horses we were unable to detect cysts or other forms of anomalies. One eye of a severely vision-impaired 18 month-old stallion, homozygous for the PMEL17 mutation was examined by light microscopy. Redundant duplication of non-pigmented ciliary body epithelium, sometimes forming cysts bulging into the posterior chamber and localized areas of atrophy in the peripheral retina were seen.
Conclusions
The MCOA syndrome is segregating with the PMEL17 mutation in the Icelandic Horse population. This needs to be taken into consideration in breeding decisions and highlights the fact that MCOA syndrome is present in a breed that are more ancient and not closely related to the Rocky Mountain Horse breed. 相似文献
Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system. 相似文献