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71.
72.
Neil C. Talbot Vernon G. Pursel Caird E. Rexroad Jr. Thomas J. Caperna Anne M. Powell Roger T. Stone 《In vitro cellular & developmental biology. Animal》1994,30(12):851-858
Summary The secondary culture of non-transformed parenchymal hepatocytes has not been possible. STO feeder cell-dependent secondary
cultures of fetal pig hepatocytes were established by colony isolation from primary cultures of 26-d fetal livers. The liver
cells had the typical polygonal morphology of parenchymal hepatocytes. They also spontaneously differentiated to form small
biliary canaliculi between individual cells or progressed further to large multicellular duct-like structures or cells undergoing
gross lipid accumulation and secretion. The secondary hepatocyte cultures expressed alpha-fetoprotein (AFP), albumin, and
β-fibrinogen mRNA, and conditioned medium from the cells contained elevated levels of transferrin and albumin. STO feeder
cell co-culture may be useful for the sustainable culture of hepatocytes from other species. 相似文献
73.
Neil R. Brandt Anthony H. Caswell Stephanie A. Lewis Carl Donald G. Ferguson Tara Brandt Jean-Pierre Brunschwig Arthur L. Bassett 《The Journal of membrane biology》1993,131(3):219-228
Summary Dyads (transverse tubule—junctional sarcoplasmic reticulum complexes) were enriched from rat ventricle microsomes by continuous sucrose gradients. The major vesicle peak at 36% sucrose contained up to 90% of those membranes which possessed dihydropyridine (DHP) binding sites (markers for transverse tubules) and all membranes which possessed ryanodine receptors and the putative junctional foot protein (markers for junctional sarcoplasmic reticulum). In addition, the 36% sucrose peak contained half of the vesicles with muscarine receptors. Vesicles derived from the nonjunctional plasma membrane as defined by a low content of dihydropyridine binding sites per muscarine receptor and from the free sarcoplasmic reticulum as defined by the Mr 102K Ca2+ ATPase were associated with a diffuse protein band (22–30% sucrose) in the lighter region of the gradient. These organelles were recovered in low yield. Putative dyads were not broken by French press treatment at 8,000 psi and only partially disrupted at 14,000 psi. The monoclonal antibody GE4.90 against skeletal muscle triadin, a protein which links the DHP receptor to the junctional foot protein in skeletal muscle triad junctions, cross-reacted with a protein in rat dyads of the same Mr as triadin. Western blots of muscle microsomes from preparations which had been treated with 100mm iodoacetamide throughout the isolation procedure showed that cardiac triadin consisted predominantly of a band of Mr 95 kD. Higher molecular weight polymers were detectable but low in content, in contrast with the ladder of oligomeric forms in rat psoas muscle microsomes. Cardiac triadin was not dissolved from the microsomes by hypertonic salt or Triton X-100, indicating that it, as well as skeletal muscle triadin, was an integral protein of the junctional SR. The cardiac epitope was localized to the junctional SR by comparison of its distribution with that of organelle markers in both total microsome and in French press disrupted dyad preparations. Immunofluorescence localization of triadin using mAb GE4.90 revealed that intact rat ventricular muscle tissue was stained following a well-defined pattern of bands every sarcomere. This spacing of bands was consistent with the interpretation that triadin was present in the dyadic junctional regions. 相似文献
74.
Neil C. Talbot Caird E. Rexroad Jr. Vernon G. Pursel Anne M. Powell Neil D. Nel 《In vitro cellular & developmental biology. Animal》1993,29(7):543-554
Summary Pig epiblast cells that had been separated from other early embryonic cells were cultured in vitro. A three-step dissection
protocol was used to isolate the epiblast from trophectoderm and primitive endoderm before culturing. Blastocysts collected
at 7 to 8 days postestrus were immunodissected to obtain the inner cell mass (ICM) and destroy trophectodermal cells. The
ICM was cultured for 2 to 3 days on STO feeder cells. The epiblast was then physically dissected free of associated primitive
endoderm. Epiblast-derived cells, grown on STO feeders, produced colonies of small cells resembling mouse embryonic stem cells.
This primary cell morphology changed as the colonies grew and evolved into three distinct colony types (endodermlike, neural
rosette, or complex). Cell cultures derived from these three colony types spontaneously differentiated into numerous specialized
cell types in STO co-culture. These included fibroblasts, endodermlike cells, neuronlike cells, pigmented cells, adipogenic
cells, contracting muscle cells, dome-forming epithelium, ciliated epithelium, tubule-forming epithelium, and a round amoeboid
cell type resembling a plasmacyte after Wright staining. The neuronlike cells, contracting muscle cells, and tubule-forming
epithelium had normal karyotypes and displayed finite or undefined life spans upon long-term STO co-culture. The dome-forming
epithelium had an indefinite life span in STO co-culture and also retained a normal karyotype. These results demonstrate the
in vitro pluripotency of pig epiblast cells and indicate the epiblast can be a source for deriving various specialized cell
cultures or cell lines. 相似文献
75.
Alan M. Goldberg John M. Frazier David Brusick Michael S. Dickens Oliver Flint Stephen D. Gettings Richard N. Hill Robert L. Lipnick Kevin J. Renskers June A. Bradlaw Robert A. Scala Bellina Veronesi Sidney Green Neil L. Wilcox Rodger D. Curren 《In vitro cellular & developmental biology. Animal》1993,29(9):688-692
Summary The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the
distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no
formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering
the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory
into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks,
a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects
and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended.
Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community—academics, industry,
and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It
is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate
scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and
refinement alternatives in toxicity testing. 相似文献
76.
77.
78.
Release of the cellular prion protein from cultured cells after loss of its glycoinositol phospholipid anchor 总被引:3,自引:0,他引:3
Borchelt David R.; Rogers Mark; Stahl Neil; Telling Glenn; Prusiner Stanley B. 《Glycobiology》1993,3(4):319-329
Secreted forms of the sialoglycoprotein designated cellularprion protein (PrPC) have been identified that cannot be explainedby alternative splicing. We report that secreted forms of PrPCderive from precursors that are bound to the plasma membraneby glycoinositol phospholipid (GPI) anchors. Secreted PrPC slowlyappeared in the culture medium of metabolically radiolabelledcells after incubations of 824 h. Digestion of nascentPrPC with phosphatidylinositol-specific phospholipase C (PIPLC)prevented the appearance of secreted PrPC. Secreted PrPc partitionedinto the aqueous phase of Triton X114 like PIPLC-releasedPrPC. While the Mr of PIPLC-released PrPC was reduced 24kDa after treatment with aqueous hydroflouric acid, which removesthe entire GPI anchor modification, the Mr of secreted PrPCwas unchanged. Both PIPLC-released and secreted PrPc were recognizedby antiserum raised against a synthetic C-terminal peptide correspondingto residues 220233 (amino acid 231 is the site of GPIattachment). We conclude that GPI-anchored PrPC is post-translationallyprocessed to remove most, if not all, of the GPI modificationand then shed into culture medium. Whether PrPC is shed afterproteolysis near the C-terminus remains to be established. Aminority of PrPC in normal Syrian hamster brain partitionedinto the aqueous phase of Triton X114 like shed PrPCsuggesting physiological significance. post-translational prion protein secretion sialoglycoprotein 相似文献
79.
80.
A series of mitochondrially inherited chloramphenicol-resistant (CAP-R) mutants were isolated in Chinese hamster cells. To determine whether the Chinese hamster CAP-R mutations were homologous to those isolated in mouse and human cell culture systems, we determined the nucleotide sequence of the region of the mitochondrial 16S rRNA gene spanning the peptidyl transferase-encoding region for eight CAP-R mutant lines in addition to the parental wildtype line. Three main conclusions are drawn from these studies. (1) Although the region of the gene encoding the peptidyl transferase domain is highly conserved relative to that of mice and rats, the contiguous sequences show less conservation. This sequence divergence not only includes the accumulation of single base pair replacements, but also the presence of small insertions or deletions. (2) For six of the CAP-R mutants, heteroplasmic single base pair changes were detected. These mapped to the same sites within the peptidyl transferase domain as the mutations found previously in mouse and human CAP-R mutants. (3) Two Chinese hamster CAP-R mutants, both with an unusual drug resistance phenotype, did not carry any mutations within the CAP-R peptidyl transferase domain. However, both carried a heteroplasmic mutation at the position corresponding to nucleotide 2505 of the mouse 16S rRNA gene, a site predicted to map within a stem/loop structure attached to this key domain of the ribosome. This is the first evidence for mitochondrial CAP-R mutations that map outside the peptidyl transferase region. 相似文献