Recent findings have suggested that the autophagic isolation membrane (IM) might originate from a domain of the endoplasmic reticulum (ER) called the omegasome. However, the morphological relationships between ER, omegasome, and IM remain unclear. In the present study, we found that hybrid structures composed of a double FYVE domain-containing protein 1 (DFCP1)-positive omegasome and the IM accumulated in Atg3-deficient mouse embryonic fibroblasts (MEFs). Moreover, correlative light and electron microscopy and immunoelectron microscopy revealed that green fluorescent protein (GFP)-tagged DFCP1 was localized on tubular or vesicular elements adjacent to the IM rims. Through detailed morphological analyses, including optimization of a fixation method and electron tomography, we observed a cluster of thin tubular structures between the IM edges and ER, part of which were continuous with IM and/or ER. The formation of these thin tubular clusters was observed in several cell lines and MEFs deficient for Atg5, Atg7, or Atg16L1 but not in FIP200-deficient cells, suggesting that they were relevant to the earlier events in autophagosome formation. Taken together, our findings indicate that these tubular profiles represent a part of the omegasome that links the ER with the IM.

The expression of MUC-1 mucin (membrane-associated mucin) and MUC-2 mucin (secretory mucin) were immunohistochemically examined in 46 invasive ductal carcinomas (IDC) and 16 intraductal papillary mucinous tumors (IPMT) of the pancreas. Intraductal papillary mucinous tumors usually reveal expansive growth. However, of the 16 IPMT examined in the present study, three showed an invasive growth pattern, which was similar to 'mucinous carcinoma', around the non-invasive growth areas. Of 46 IDC, MUC-1 mucin detected by monoclonal antibodies, DF3 and MY.1E12, was expressed in 44 cases (96%) and in 45 cases (98%), respectively, whereas MUC-2 mucin detected by polyclonal antibody, anti-MRP, was not expressed in any of the cases (0%). In contrast, in the non-invasive growth areas of the 16 IPMT, MUC-1 mucin detected by DF3 and MY.1E12 was expressed in four cases (25%) and in six cases (38%), respectively, whereas MUC-2 mucin detected by anti-MRP was expressed in 13 cases (81%). The invasive growth areas of the three IPMT showed positive expression of MUC-1 mucins detected by DF3 and MY.1E12, although the non-invasive growth areas showed negative expression of MUC-1 mucins, except for their focal positive expression in one of the three cases. These findings indicate that the invasive growth areas of IPMT acquire a characteristic of MUC-1 mucin expression that is usually seen in IDC.

The homeobox gene Six4/AREC3 is a member of the vertebrate Six family of transcription factor genes. In this study we describe the cloning and expression of three zebrafish homologues of Six4/AREC3, six4.1, six4.2 and six4.3. These zebrafish Six4 proteins show high homology to the mammalian Six4/AREC3 proteins in the most C-terminal region in addition to the Six domain and homeodomain. Whole-mount in situ hybridization showed that six4.1 is expressed in the presumptive cranial placodal precursor cells, and later in the olfactory, otic and lateral line placodes. six4.2 is expressed in the presomitic mesoderm, somites and pectoral fin bud. six4.3 appears to be a unique member of the Six4 proteins and is expressed ubiquitously at gastrulation and later in the tectum.

Development of a new monoclonal antibody (mAb) MY.1E12 which reacts with sialylated MUC1 mucins is described. The mAb did not react with any component in the lysates of COS‐1 cells, whereas it bound to sialylated MUC1 mucins produced by COS‐1 cells transiently transacted with MUC1 mucin cDNA, strongly suggesting that the expression of the epitope of mAb MY.1E12 depends on the presence of the MUC1 mucin core peptide. The requirement of sialyl residues for antibody recognition was established by Western blotting analysis of extracts of various carcinoma cells and in situ desialylation. In all cases, the mAb binding of electrophoretically separated MUC1 mucin diminished after desialylation by mild acid hydrolysis. When Capan‐1 pancreatic carcinoma cells were pretreated with benzyl‐JV‐acetylgalactosaminide in culture, the MUC1 mucins produced under these conditions, which were detected by core peptide‐specific mAbs, did not react with mAb MY.1E12. These results suggest that 0‐linked carbohydrate chains are important for the mAb binding.

Surgical specimens of the normal kidney and of renal cell carcinoma (RCC) tissues at different stages of progression and of various histological grades were examined for the expression of MUC1 mucins with sialylated carbohydrates (sialylated MUC1 mucins) using a monoclonal antibody MY.1E12. Immunohistochemical studies revealed that the binding sites for this antibody were localized to the apical side of the epithelial cells of the distal convoluted tubules, Henle's loops and collecting ducts. However, proximal convoluted tubules, where RCC is considered to originate, were not stained. This antibody also bound strongly to RCC at advanced stages of progression and at metastatic sites, and to RCC of histologically high grades (undifferentiated). The epitope, presumably sialylated MUC1 mucin, was detected not only along the surface of the cell membranes but also in the cytoplasm. The level of expression of sialylated MUC1 mucins was inversely correlated with the survival of the patients with RCC and the disease-free survival period after curative surgery. Western blot analysis demonstrated that the electrophoretic mobility of sialylated MUC1 mucins of RCC was greater than that from the normal kidney. It is suggested that high levels of expression of sialylated MUC1 mucins in certain human RCC populations correlate with the aggressiveness of the disease, such as the tendency to form metastasis. © 1999 Cancer Research Campaign