PubMed · 10563306
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E Wang, A H Merrill. 2000. Ceramide synthase.. https://doi.org/10.1016/s0076-6879(00)11062-6
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Human surfactant protein C (hSP-C) is synthesized by the alveolar type 2 cell as a 197 amino acid integral membrane proprotein and proteolytically processed to a secreted 3.7 kDa mature form. Although the SP-C null mouse possesses a non-lethal phenotype, a heterozygous substitution of A for G in the first base of intron 4 of the human SP-C gene (c.460+1A>G) has been reported in association with familial interstitial lung disease and absence of mature protein. This mutation produces a splice deletion of exon 4 (deltaExon4) resulting in removal of a positionally conserved cysteine in the C-terminal flanking propeptide. Based on a prior study showing that an identical deletion in the rat isoform diverted mutant protein to stable aggregates, we hypothesized that expression of the deltaExon4 mutation would result in disruption of intracellular trafficking of both mutant and wild-type proSP-C. We tested this in vitro using fusion proteins of EGFP conjugated either to wild-type SP-C (EGFP/hSP-C(1-197)) or to SP-C deleted of Exon4 (EGFP/hSP-C(deltaExon4)). Fluorescence microscopy showed that EGFP/hSP-C(1-197) transfected into A549 cells was expressed in a punctuate pattern in CD63 (+) cytoplasmic vesicles, whereas EGFP/hSP-C(deltaExon4) accumulated in ubiquitinated perinuclear inclusions linked to the microtubule organizing center. A similar juxtanuclear pattern was observed following transfection of SP-C cDNA lacking only cysteine residues in the C-terminal propeptide encoded by Exon 4 (EGFP/hSP-C(C120/121G)). To evaluate whether mutant proSP-C could function as a dominant negative, EGFP/hSP-C(deltaExon4) was cotransfected with HA-tagged hSP-C(1-197) and resulted in the restriction of both forms to perinuclear compartments. Addition of Na(+) 4-phenylbutyrate, a facilitator of trafficking of other misfolded proteins, attenuated the aggregation of EGFP/hSP-C(deltaExon4). We conclude that c.460+1A>G mutation of human SP-C results in disruption of disulfide-mediated folding encoded by Exon 4 leading to diversion of unprocessed proSP-C to aggresomes. The heterotypic oligomerization of hSP-C(1-197) and hSP-C(deltaExon4) provides a molecular mechanism for the dominant-negative effect observed in vivo.
In eukaryotes, poly(A) tails usually act as stabilizers of intact mRNAs, whereas in E. coli they serve to accelerate the destruction of fragments. The mechanisms underlying these contrasting effects of the same RNA modification are discussed.
BACKGROUND: The number of microalgal strains known to date is enormous and continuously growing, and their characterization accordingly requires quick and reliable methodologies. METHODS: Asynchronously growing logarithmic (3- and 6-day cultures) and stationary (9-day cultures) phase cell populations of two algae species that are difficult to distinguish microscopically (one Chlorella sp., C. vulgaris [c-27], and another that might belong to the same genus, SA-3 algae exsymbiotic from Paramecium bursaria) were characterized by means of flow cytometry (FCM). Forward light scatter (FSC) of algae was monitored in association with their 90 degrees side light scatter (SSC) and fluorescence of endogenous chlorophyll (FL3-height). RESULTS: Two-parameter FSC versus SSC and FSC versus FL3-height plots distinctly showed growth-specific compartmentation of algae into discrete cell subpopulations staying at a particular stage of the life cycle, and numbers of cells constituting these subpopulations could be quantitated. The growth pattern of C. vulgaris (c-27) differed substantially from that of SA-3 algae, particularly in the late-logarithmic (6-day) cultures. At this phase of growth, C. vulgaris (c-27) cells compartmentalized into three subpopulations, whereas SA-3 cells compartmentalized into two subpopulations. Different compartmentations of optical signals from late-logarithmic phase SA-3 algae and C. vulgaris (c-27) likely were caused by the differences in timing of the life cycle stages of these types of cells. CONCLUSIONS: Growth-specific compartmentation of vegetating microalgae by FCM provides a good basis for characterization of morphologically similar algae species. Because algae are also present in symbiotic relationships with other organisms, this tool might be of potential interest for the study of symbiosis mechanisms.