Abstract:
:A method is presented for detection of lysosomal hydrolase activity in situ in colonies of Chinese hamster ovary cells. This method was used to screen for mutants deficient in lysosomal alpha-mannosidase. Mutagenized cells were replicated onto filter papers [Esko, J. D. & Raetz, C. R. H. (1978) Proc. Natl. Acad. Sci. USA 75, 1190-1193] and allowed to divide for 11-14 days; cells on the filter replicas were lysed, and the filters were incubated with 4-methylumbelliferyl-alpha-D-mannopyranoside at pH 4.4 and then photographed under UV light. Colonies for which the replicas exhibited little or no fluorescence were picked from the master plates and purified; mutants with decreased alpha-mannosidase activity were obtained at a frequency of 1 per 1500 mutagenized cells. Analysis of one of these mutants showed that lysosomal alpha-mannosidase activity was 18% of that from wild-type cells, whereas the activities of alpha-mannosidases not of lysosomal origin were similar in the wild type and mutant (these included both Golgi-associated and cytosolic enzymes as well as a novel acid alpha-mannosidase seen only in cells from confluent culture). The mutant contained normal levels of other lysosomal hydrolases. Both wild-type and mutant cells secreted alpha-mannosidase into the medium at levels proportional to those found inside the cells. Incubation of mutant cells with secretions from the wild type resulted in partial restoration of enzyme activity. Added enzyme was localized in the lysosomal fractions; uptake of added enzyme was inhibited by mannose 6-phosphate and fructose 1-phosphate, which are known to inhibit uptake of lysosomal enzymes into human diploid fibroblasts.
journal_name
Proc Natl Acad Sci U S Aauthors
Robbins ARdoi
10.1073/pnas.76.4.1911subject
Has Abstractpub_date
1979-04-01 00:00:00pages
1911-5issue
4eissn
0027-8424issn
1091-6490journal_volume
76pub_type
杂志文章abstract::Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analy...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1119351109
更新日期:2012-01-10 00:00:00
abstract::Eukaryotic cells require N-linked carbohydrates for survival. However, the biosynthetic intermediate Man5GlcNAc2Asn, in place of mature N-linked structures, allows glycoprotein synthesis and somatic cell growth to proceed normally. To determine whether the same would be true in a complex biological situation, the gene...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.91.2.728
更新日期:1994-01-18 00:00:00
abstract::Na(+)-channel inactivation is proposed to occur by binding of an intracellular inactivation gate to a hydrophobic inactivation gate receptor in the intracellular mouth of the pore. Amino acid residues in transmembrane segment S6 of domain IV (IVS6) that are critical for fast inactivation were identified by alanine-sca...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.91.25.12346
更新日期:1994-12-06 00:00:00
abstract::Paleoclimate studies suggest that increased global warmth during the Eocene epoch was greatly amplified at high latitudes, a state that climate models cannot fully reproduce. However, proxy estimates of Eocene near-Antarctic sea surface temperatures (SSTs) have produced widely divergent results at similar latitudes, w...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1321441111
更新日期:2014-05-06 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1508990112
更新日期:2015-06-30 00:00:00
abstract::B-1a cells reside in both the peritoneal cavity and the spleen. LPS stimulates splenic B-1a to differentiate to plasma cells producing natural IgM specific for microbial and self antigens. However, there are conflicting views as to whether the B-1a cells divide before this differentiation occurs, and hence how the res...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0700001104
更新日期:2007-03-13 00:00:00
abstract::Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 iso...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.90.11.5297
更新日期:1993-06-01 00:00:00
abstract::Regulated proteolysis is required to execute many cellular programs. In Caulobacter crescentus, timely degradation of the master regulator CtrA by ClpXP protease is essential for cell-cycle progression and requires the colocalization of CtrA and RcdA. Here, we establish a biochemical framework to understand regulated ...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0701776104
更新日期:2007-04-17 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1217361110
更新日期:2013-02-12 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.92.2.626
更新日期:1995-01-17 00:00:00
abstract::Downstream placement of a strong transcriptional termination signal has made possible the cloning of bacteriophage T5 promoters known to exhibit high signal strength. The cloning system constructed contains two easily assayable indicator functions whose expression is controlled by the integration of promoters and term...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.78.8.4936
更新日期:1981-08-01 00:00:00
abstract::We have screened a human DNA library using as probe a chimpanzee sequence that contains homology to the polymerase gene of the endogenous baboon virus. One set of overlapping clones spans about 20 kilobases and contains regions of DNA sequence homology to the gag p30, gag p15, and polymerase genes of Moloney murine le...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.79.15.4709
更新日期:1982-08-01 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.251670699
更新日期:2002-05-14 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1733335100
更新日期:2003-09-16 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0509635102
更新日期:2005-12-27 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.90.19.9036
更新日期:1993-10-01 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0601075103
更新日期:2006-08-22 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.97.16.9064
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
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更新日期:2006-12-19 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.91.14.6433
更新日期:1994-07-05 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0901319106
更新日期:2009-09-15 00:00:00
abstract::An in vitro system [Weil, P. A., Luse, D. S., Segall, J. & Roeder, R. G. (1979) Cell 18, 469-484 and Manley, J. L., Fire, A., Cano, A., Sharp, P. A. & Gefter, M. L., (1980) Proc. Natl. Acad. Sci USA 77, 3855-3859] was adapted for studying initiation of transcription of the ovalbumin gene. The DNA template was a cloned...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.78.2.879
更新日期:1981-02-01 00:00:00
abstract::Germ-line mutations of the BRCA2 tumor suppressor gene greatly increase the risk of developing breast and ovarian cancers. Here, we show that wild-type BRCA2, but not a tumor-specific truncated mutant BRCA2, synergizes with the nuclear receptor coactivator p160 GRIP1 to enhance transcriptional activation by androgen r...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1132020100
更新日期:2003-06-10 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.88.12.5312
更新日期:1991-06-15 00:00:00
abstract::The N-methyl-d-aspartate (NMDA) glutamate receptor (NMDAR), long implicated in developmental plasticity, shows decay time kinetics that shorten postnatally as NR2A subunits are added to the receptor. Neither the mechanism nor immediate effect of this change is known. We studied developing NMDAR currents by using visua...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0335786100
更新日期:2003-02-04 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.1015622108
更新日期:2011-04-26 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.83.13.4764
更新日期:1986-07-01 00:00:00
abstract::To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protei...
journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.0912989107
更新日期:2010-06-22 00:00:00
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journal_title:Proceedings of the National Academy of Sciences of the United States of America
pub_type: 杂志文章
doi:10.1073/pnas.95.18.10949
更新日期:1998-09-01 00:00:00