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We have developed a technique for the simultaneous chromosomal assignment of multiple human DNA sequences from DNA fingerprints obtained by the arbitrarily primed polymerase chain reaction (AP-PCR). Radioactively labeled human AP-PCR products are hybridized to DNA fingerprints generated with the same arbitrary primer from human/rodent monochromosome cell hybrids after electroblotting to a nylon membrane. Human-specific hybridization bands in the human/rodent fingerprints unambiguously determine their chromosome of origin. We named this method simultaneous hybridization of arbitrarily primed PCR DNA fingerprinting products (SHARP). Using this approach, we determined the chromosomal origins of most major bands of human AP-PCR fingerprints obtained with two arbitrary primers. Altogether, the chromosomal localization of near 50 DNA fragments, comprehensive of all human chromosomes except chromosomes 21 and Y, was achieved in this simple manner. Chromosome assignment of fingerprint bands is essential for molecular karyotyping of cancer by AP-PCR DNA fingerprinting. The SHARP method provides a convenient and powerful tool for this purpose.

We have previously described the abnormal localization of resident Golgi proteins and O-glycans in the rough endoplasmic reticulum of mucin-secreting HT-29 M6 colon cancer cells, suggesting altered protein trafficking in these cells [Egea, Franci~A, Gambús, Lesuffleur, Zweibaum and Real (1993) J. Cell Sci. 105, 819–830]. In the present work, we have chosen lysosomal α-glucosidase as a reporter to examine the intracellular traffic of glycoproteins in M6 cells. We have compared the synthesis and processing of α-glucosidase in mucin-secreting M6 cells and in Caco-2 colon cancer cells, the latter resembling normal absorptive intestinal epithelium. Our results show that α-glucosidase processing and secretion is markedly delayed in M6 cells as compared with Caco-2 cells or normal fibroblasts, and this delay is caused by an accumulation of α-glucosidase precursor form in the trans-Golgi network. Furthermore, treatment of Caco-2 cells with brefeldin A led to changes in α-glucosidase maturation similar to those observed in untreated M6 cells. To determine whether altered processing occurs in other cultured cells, a panel of cancer cell lines and cultures from normal exocrine pancreas were examined. In pancreas-derived cultures, α-glucosidase showed a processing pattern different from that described until now. Only HT-29 cells and HT-29-derived subpopulations displayed a defect in α-glucosidase maturation. In conclusion, α-glucosidase processing is more diverse than has previously been described; this finding may have tissue-specific functional implications.

Glucocorticoids play a key role in mediating stress responses in vertebrates. Corticosterone (CORT) is the main glucocorticoid produced in amphibians, birds, and reptiles, and regulates several metabolic functions. The most common methods for quantifying CORT are competitive binding immunoassays: radioimmunoassay (RIA) and enzyme immunoassay (EIA). RIA has been broadly used since the 1980’s but it requires radioactivity. Commercial EIA kits permit quantifying hormone levels without radioactivity although the requirement for a larger sample volume may be a strong limitation for measurements involving larval amphibians. Here we used Xenopus laevis tadpoles to compare the performance of three commonly used procedures for determination of CORT: RIA on a chloroform extract of whole-body homogenate, EIA on plasma, and EIA on supernatant of whole-body homogenate. We treated tadpoles with exogenous CORT at 0, 25, 50, and 100 nM. RIA could distinguish between 0 and 25 nM, and EIA on plasma between 0 and 50 nM, whereas whole-body homogenate EIA only detected significant differences between 0 and 100 nM. Each procedure presents advantages and disadvantages regarding sensitivity, the use of radioactivity, sample size, handling time, and economic cost. RIA is preferred when studying small-bodied animals from which blood samples cannot be obtained. When CORT level differences are intermediate and blood sampling is possible, EIA on plasma is a good non-radioactive alternative. EIA on whole-body homogenates may be useful to assess qualitative changes in CORT levels when considerable differences are expected. Finally, we discuss our findings in the context of previous studies on CORT in amphibians.