Mammary Stat5 abundance and activity are not altered with lactation state in cows.

Stat5 is a key intracellular mediator of prolactin signalling and can activate transcription of milk proteins in response to prolactin. Therefore, in animals such as mice where lactation is dependent on prolactin, Stat5 is likely to play an important role in establishing or maintaining lactation in the mammary gland. However, little is known about its role in lactation in the dairy cow. In order to address this, the levels of Stat5a and Stat5b protein, mRNA and Stat5 DNA-binding activity were measured in mammary tissue from mice and cows at different lactational states. In the cow, Stat5a and Stat5b protein and mRNA levels, as well as Stat5 DNA-binding activity were unaltered between pregnancy and established lactation. In contrast, in the mouse Stat5a and Stat5b protein, as well as Stat5 DNA-binding activity were clearly increased during lactation whereas Stat5a and Stat5b mRNA levels were highest during pregnancy as has been previously described. In both species only a minority of the epithelial cell nuclei were Stat5 positive during established lactation. These results suggest that there are significant differences in the biological role of Stat5 in controlling lactation between ruminants and rodents.

Elevation of lactoferrin gene expression in developing, ductal, resting, and regressing parenchymal epithelium of the ruminant mammary gland.

Accumulation of lactoferrin mRNA in mammary tissue from virgin, pregnant, lactating, and involuting ewes and cows was localized using 35S-labeled cRNA probes. Expression of lactoferrin was low in the glands of virgin animals. In the glands of animals in early pregnancy, very high expression occurred in the ducts and immature alveoli, but expression tended to decrease as the alveoli matured. In the lactating and involuting gland, expression was generally low or absent in actively secreting alveoli and high in alveoli that had an accumulation of vesicles in the lumen and secretory epithelium, which was indicative of stasis. Occasionally, expression of lactoferrin was seen in cells that appeared to be secretory, particularly in involuting glands. Lactoferrin mRNA was expressed not only at different sites from other milk protein genes, such as alpha-lactalbumin and alpha s1-casein, but also during different stages of mammary development, supporting the view that the expression of lactoferrin is regulated differently from that of other milk proteins. For all ewes and cows, lactoferrin mRNA was detected in the epithelial ducts of the mammary parenchyma and the teat in a gradient that increased in ducts nearer the teats. The expression of lactoferrin in the ductal epithelium close to the teat was consistent with the antibacterial role of lactoferrin.

Interstitial collagenase gene expression in colonic neoplasia.

Tumor invasion and metastasis are complex phenomena believed to be facilitated by the disruption of collagen and elastin fibers in the extracellular matrix. Interstitial collagenase gene expression was studied in colonic adenocarcinoma and adenoma using in situ hybridization. The data indicated that three cell types within the tumor stroma expressed collagenase transcripts; they were eosinophils, fibroblasts, and vascular endothelium. In all 12 adenocarcinomas, a high to moderate level of expression was seen in 1 to 5% of eosinophils and in occasional fibroblasts, whereas these cell types in non-neoplastic mucosa adjacent to tumor showed no detectable expression. Two adenocarcinomas showed expression in hyperplastic endothelium in vascularized granulation tissue. Two out of three adenomas showed expression in eosinophils and fibroblasts at a reduced level. Tissue inhibitor of metalloproteinase-1 gene expression was, however, negligible in all tissue examined. These results suggest that interstitial collagenase gene activation in the tumor stroma, especially eosinophils, may have an important role in tumor invasion and metastasis.

Rapid beta-lactoglobulin genotyping of cattle using the polymerase chain reaction.

A polymerase chain reaction (PCR) assay has been developed for genotyping beta-lactoglobulin A and B variants in dairy cattle. Either blood or semen samples can be used as a source of DNA. The method is accurate, faster than Southern blot analyses and should prove a useful tool in breeding programmes.

Association of degradation and secretion of three chimeric polypeptides in Escherichia coli.

We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.