Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts

Summary Total insoluble collagen and hydroxyproline formation were examined in lung embryonic fibroblasts (IMR-90) grown in the presence or absence of added ascorbate. As expected, when the cells from both groups (+ and −ascorbate) are pulsed with [¹⁴C]proline in the presence of ascorbate, the percent hydroxylation in a 24-hr period does not vary significantly. However, there are dramatic differences in the quantity and quality of the insoluble collagen fraction produced by those cells grown for a long period of time with added ascorbate. Those cells deprived of continuous addition of ascorbate to the culture medium do not display large quantities of accumulated collagen in the cell layer fractions as measured by the hydroxyproline content, whereas the cells grown in the presence of ascorbate contain significant amounts of accumulated collagen. A new method for examining the extracellular insoluble collagen produced in cell cultures is described in these studies. With the aid of pancreatic elastase relatively pure insoluble collagen can be obtained from cells grown in culture. In those cells grown in the presence of ascorbate, the purified insoluble collagen yeilds appropriately banded fibrils when examined in the electron microscope and has an amino-acid composition that is compatible with pure collagen. On the other hand, those cells grown in the absence of ascorbate do not yield purified insoluble collagen as determined by these same criteria. The elastase procedure for the purification of insoluble collagen in cell cultures is simple, easy to use and allows one to assess additional aspects of collagen biosynthesis.     

A role for natural killer T cells in asthma

In several mouse models, natural killer T cells have recently been found to be required for the development of airway hyper-reactivity, a cardinal feature of asthma. Moreover, in patients with chronic asthma, natural killer T cells with a T-helper-2-like phenotype (that is, that express CD4 and produce T helper 2 cytokines) are present in the lungs in large numbers. In this Opinion article, we suggest that natural killer T cells, which express a restricted T-cell receptor and respond to glycolipids rather than protein antigens, have a previously unsuspected but crucial role, distinct from that of T helper 2 cells, in the pathogenesis of asthma.     

Primary structure-based function characterization of BRCT domain replicates in BRCA1

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.     

Amelogenin cross-amplification in the family Bovidae and its application for sex determination

Sex-specific sequence variability of the amelogenin gene had been observed in a variety of mammalian species. In our study, the suitability of the amelogenin gene for sex determination in different species of the family Bovidae was examined. Based on a sequence insertion/deletion characteristic for X- and Y-specific amelogenin (AMELX and AMELY), PCR amplification on male and female genomic DNA from domestic and wild bovine species, sheep and goat, consistently displayed a sex-specific pattern. Thus, the amelogenin amplification by PCR proved to be a reliable method for sex determination not only in domestic and wild species of the tribe Bovini, but also in the related species sheep and goat. Sex determination using the amelogenin-based assay can be performed with at least 40 pg of genomic DNA. The assay enables the investigation of small amounts of DNA from meat, hair, bones, and embryo biopsies to identify species and sex for a number of applications in animal production, forensics, population research, and monitoring within the family Bovidae. Sequence comparison of the amplified amelogenin gene region specific for male and female animals from domestic and wild bovide species revealed further sequence variations within and between sexes as well as between species. Sequence variations in the AMELX gene can be applied to discriminate Bos and Bison individuals from other bovine species, and also from sheep and goat.     

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.