Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection

Polygalacturonase-inhibiting protein (PGIP) is a cell wall-associated protein that specifically binds to and inhibits the activity of fungal endopolygalacturonases. The Phaseolus vulgaris gene encoding PGIP has been cloned and characterized. Using a fragment of the cloned pgip gene as a probe in Northern blot experiments, it is demonstrated that the pgip mRNA accumulates in suspension-cultured bean cells following addition of elicitor-active oligogalacturonides or fungal glucan to the medium. Rabbit polyclonal antibodies specific for PGIP were generated against a synthetic peptide designed from the N-terminal region of PGIP; the antigenicity of the peptide was enhanced by coupling to KLH. Using the antibodies and the cloned pgip gene fragment as probes in Western and Northern blot experiments, respectively, it is shown that the levels of PGIP and its mRNA are increased in P. vulgaris hypocotyls in response to wounding or treatment with salicylic acid. Using gold-labeled goat-anti-rabbit secondary antibodies in EM studies, it has also been demonstrated that, in bean hypocotyls infected with Colletotrichum lindemuthianum, the level of PGIP preferentially increases in those cells immediately surrounding the infection site. The data support the hypothesis that synthesis of PGIP constitutes an active defense mechanism of plants that is elicited by signal molecules known to induce plant defense genes.     

Sporulation: an alternative way to recover recombinant proteins from Bacillus subtilis

A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin-1 receptor antagonist (IL-Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step. (c) 1995 John Wiley & Sons, Inc.     

Immunochemical studies of the inactivation of aspartate transcarbamylase by stationary phase Bacillus subtilis cells. Evidence for selective, energy-dependent degradation.

The aspartate transcarbamylase of Bacillus subtilis is stable in exponentially growing cells, but undergoes rapid, energy-dependent inactivation when growth is inhibited by nutrient depletion or addition of antibiotics or other inhibitors of metabolism. This inactivation has been analyzed by a variety of immunochemical techniques, including direct and indirect immunoprecipitation of extracts of cells labeled with 3H-amino-acids, microcomplement fixation, and neutralization of enzymatic activity. The ability of the antibody preparation to react with various denatured, chemically modified, and proteolytically degraded forms of aspartate transcarbamylase was demonstrated. All of the techniques showed that cross-reactive protein disappeared from the cells at the same rate as enzymatic activity, and that little or no immunoprecipitable material of lower than native molecular weight was detectable during inactivation. The disappearance of material cross-reactive with aspartate transcarbamylase occurred prior to the increase in protein degradation that normally occurs in stationary B. subtilis cells and proceeded at a rate at least 20 times greater than general protein degradation. The rate of disappearance was unaffected in mutant strains deficient in intracellular protease activity or in cells treated with inhibitors of protein turnover. Aspartate transcarbamylase was shown to be stable in growing cells. We conclude that the inactivation of aspartate transcarbamylase in vivo involves, or is rapidly followed by, selective, energy-dependent degradation of the protein by a system that appears to involve a previously undescribed protease of B. subtilis.     

A histochemical study of the binding of 125I-HCG to the rat ovary throughout the estrous cycle

Summary Changes in the distribution of the in vitro uptake of ¹²⁵I-HCG by the ovaries of adult rats were examined histochemically throughout the estrous cycle.Only in follicles wider than 500 m, occurring mainly at diestrus and proestrus, could granulosa cells bind the labelled hormone. The labelling increased with follicular size and decreased in intensity from the peripheral granulosa cells inwards. No uptake occurred in the oocytes, in the cells of the cumulus oophorus nor in the granulosa cells of the atretic follicles.The binding capacity of the newly-formed corpora lutea of estrus was less than that of preovulatory follicles. The uptake of ¹²⁵I-HCG by corpora lutea during the first cycle reached its maximum at diestrus but fell sharply by proestrus. The uptake was patchy in the corpora lutea of the second cycle and not significant in the older ones.The uptake of ¹²⁵I-HCG by thecae increased with follicular size and was greater in the thecae of atretic follicles than in the thecae of growing follicles of like size. There was a greater uptake in the last formed interstitial tissue than there was in older tissue.At proestrus, the uptake of ¹²⁵I-HCG was unaffected by the LH surge at 18.00h but had decreased slightly at 24.00 h.The implications of these data in relation to the regulation of receptor sites, is discussed.     

Changes in cellular gene expression in rat thyroid epithelial cells transformed by the Kirsten murine sarcoma virus

We have exploited a recently characterized system of rat thyroid epithelial cells transformed by the wild-type (wt) and a temperature-sensitive (ts) mutant strain of the Kirsten murine sarcoma virus (Ki-MSV) in order to study the effects of the K-ras oncogene on the gene expression of differentiated thyroid epithelial cells. By using cDNAs isolated from normal thyroid glands as probes, we were able to identify three sets of cellular sequences whose expression is influenced by the v-K-ras oncogene. The first set of genes is irreversibly repressed by transformation with both the wt and the ts viruses. The second set of genes is repressed in the ts-Ki-MSV-transformed cells but not in the same cells grown at the nonpermissive temperature. A third set of genes is present at higher levels at the nonpermissive temperature than at the permissive temperature. This system has allowed us to isolate and characterize a number of cDNA clones belonging to each of these three sets of genes. These specific cDNAs are suitable probes to study phenotypical changes during transformation of epithelial cells.     

Biomarker of food intake for assessing the consumption of dairy and egg products

Dairy and egg products constitute an important part of Western diets as they represent an excellent source of high-quality proteins, vitamins, minerals and fats. Dairy and egg products are highly diverse and their associations with a range of nutritional and health outcomes are therefore heterogeneous. Such associations are also often weak or debated due to the difficulty in establishing correct assessments of dietary intake. Therefore, in order to better characterize associations between the consumption of these foods and health outcomes, it is important to identify reliable biomarkers of their intake. Biomarkers of food intake (BFIs) provide an accurate measure of intake, which is independent of the memory and sincerity of the subjects as well as of their knowledge about the consumed foods. We have, therefore, conducted a systematic search of the scientific literature to evaluate the current status of potential BFIs for dairy products and BFIs for egg products commonly consumed in Europe. Strikingly, only a limited number of compounds have been reported as markers for the intake of these products and none of them have been sufficiently validated. A series of challenges hinders the identification and validation of BFI for dairy and egg products, in particular, the heterogeneous composition of these foods and the lack of specificity of the markers identified so far. Further studies are, therefore, necessary to validate these compounds and to discover new candidate BFIs. Untargeted metabolomic strategies may allow the identification of novel biomarkers, which, when taken separately or in combination, could be used to assess the intake of dairy and egg products.