Diaphragmatic pathology: a cause of clinically unexplained death in the perinatal/paediatric age group.

Sudden unexpected death in infancy and childhood requires a 'full' post-mortem investigation. Guidance from the Royal College of Pathologists recommends sampling of all the major organs. However, the diaphragm does not feature in this or in most lists of routine histology. Our aim is to emphasize the importance of sampling the diaphragm for histological examination during autopsy. We describe three autopsy cases of clinically unexplained death in the perinatal and paediatric age group that showed significant pathology of the diaphragm. In Case 1, a previously healthy five-year-old girl collapsed suddenly and died four days later. In Case 2, an eight-month-old infant had repeated episodes of respiratory arrest that culminated in death. Autopsy demonstrated a predominantly diaphragmatic myositis. In Case 3 a female neonate had a respiratory arrest three days after birth and died less than a month later. Autopsy showed multiple large calcified necrotic fibres in the diaphragm. The diaphragm is seldom sampled at autopsy. In the first two cases a predominantly diaphragmatic myositis was either the direct or underlying cause of death. In the third case long-standing diaphragmatic pathology of uncertain cause may have contributed to the original respiratory arrest. Had the diaphragm not been examined histologically, the cause of death would have remained unascertained in these cases. In cases of sudden death in infancy and childhood, failure to reach a diagnosis may lead to undue suspicion falling upon the child's carers. This underscores the need for full histology at post-mortem in child deaths, including diaphragmatic sampling.

Competition between nuclear localization and secretory signals determines the subcellular fate of a single CUG-initiated form of FGF3.

The presumed open reading frame for mouse FGF3, starting at the most 5' AUG codon, predicts a hydrophobic N-terminus characteristic of a signal peptide for secretion. However, in reticulocyte lysates and transfected COS-1 cells, the full-length Fgf-3 cDNA is translated almost exclusively from an upstream CUG codon. The resultant products are distributed in both the nucleus and the secretory pathway, implying that the single CUG-initiated form of FGF3 has dual fates. By analysing a series of deletion and replacement mutants and by linking parts of FGF3 to a heterologous protein, we show that secretion is mediated by cleavage adjacent to the previously defined signal peptide, whereas nuclear localization is determined primarily by a classical but relatively weak bipartite motif. In the context of FGF3, nuclear localization also requires the N-terminal sequences which lie upstream of the signal peptide. Thus, the subcellular fate of FGF3 is determined by the competing effects of signals for secretion and nuclear localization within the same protein, rather than by alternative initiation or processing.

Subcellular fate of the int-2 oncoprotein is determined by choice of initiation codon.

Fibroblast growth factors (FGFs) have been implicated in many aspects of cell growth and differentiation both in normal and neoplastic settings. For example, the mouse int-2 gene, which encodes an FGF-related product, is a frequent target of proviral activation in carcinomas induced by mouse mammary tumour virus, but apparently functions at discrete stages of normal embryonic development. Six classes of int-2 messenger RNA have been identified in embryonic cells, each of which is predicted to encode the same 245-amino-acid protein. But all known int-2 transcripts include sequences upstream of the AUG codon presumed to be the initiation codon. Here we report an additional N-terminally extended int-2 gene product initiated at an in-frame CUG codon. In COS-1 cells transiently transfected with appropriate int-2 complementary DNAs, the AUG-initiated product is found predominantly in the secretory pathway, whereas the CUG-initiated form is localized to the nucleus. These data indicate that the Int-2 oncoprotein could influence cellular behaviour by two distinct mechanisms.

Int-2: a member of the fibroblast growth factor family has different subcellular fates depending on the choice of initiation codon.

The int-2 gene, which encodes a member of the fibroblast growth factor family, was discovered as a protooncogene transcriptionally activated following proviral insertion into adjacent chromosomal DNA. Analyses of the synthesis and processing of the int-2 protein, using an SV40-based vector to express cloned cDNA, showed four major products in the size range 27.5-31.5 kd that were associated with the secretory pathway. Further experiments using a cell-free translation system programmed with int-2 cRNA revealed a larger N-terminally extended protein. Site-directed mutagenesis of possible initiation codons confirmed that the first in-frame AUG codon would specify the start of a protein that includes a signal peptide for transport into the endoplasmic reticulum. However, protein synthesis also initiates at an upstream CUG codon to yield a polypeptide extended at the N-terminus by 29 amino acids. Immunofluorescent staining showed that a substantial proportion of the CUG-initiated protein resides in the cell nucleus, while a truncated int-2, lacking both the N-terminal extension and the signal peptide, was exclusively nuclear. These observations suggest that a nuclear localisation signal occurs in the body of the int-2 molecule, but is only accessible to the nuclear transport system if entry to the secretory pathway is compromised. Thus, the choice of initiation codon changes the subcellular fate of the int-2 protein and provides the potential for a duality of function through alternative transport pathways.

Characterization of int-2: a member of the fibroblast growth factor family.

int-2 was discovered as a proto-oncogene transcriptionally activated by MMTV proviral insertion during mammary tumorigenesis in the mouse. Sequence analysis showed int-2 to be a member of the fibroblast growth factor family of genes. In normal breast and most other adult mouse tissues, int-2 expression was not detected except for low levels in brain and testis. However, using in situ hybridization, expression was found at a number of sites during embryonic development, from day 7 until birth. An analysis of the int-2 transcripts found in embryonal carcinoma cells revealed six major classes of RNA initiating at three promoters and terminating at either of two polyadenylation sites. Despite the transcriptional complexities, all size classes of RNA encompass the same open reading frame. Using an SV40 early promoter to drive transcription of an int-2 cDNA in COS-1 cells, several proteins were observed. These were shown to be generated by initiation from either of two codons: One, a CUG, leads to a product which localizes extensively to the cell nucleus and partially to the secretory pathway. In contrast, initiation at a downstream AUG codon results in quantitative translocation across the endoplasmic reticulum and the accumulation of products ranging in size from 27.5 x 10(3) Mr to 31.5 x 10(3) Mr in organelles of the secretory pathway. These proteins represented glycosylated and non-glycosylated forms of the same primary product with or without the signal peptide removed. These findings suggest the potential for a dual role of int-2; an autocrine function acting at the cell nucleus, and a possible paracrine action through a secreted product.

Detection and characterization of the fibroblast growth factor-related oncoprotein INT-2.

Products of the fibroblast growth factor-related proto-oncogene int-2 have been detected by using a monoclonal antibody and polyclonal antisera raised against synthetic peptides predicted from the DNA sequence. COS-1 monkey cells transfected with int-2 DNA linked to the simian virus 40 early promoter contained at least four int-2-specific proteins, presumably representing modified forms of the expected 27-kilodalton primary translation product. The level of expression was increased approximately six- to eightfold by mutation of sequences around the presumed initiation codon, negating their capacity to encode a short oligopeptide in the +1 reading frame. Both tunicamycin inhibition and in vitro translation experiments indicated that some of the modifications correspond to asparagine-linked glycosylation, for which the sequence predicts a single site. In line with the similarities between INT-2 and other fibroblast growth factors, the in vitro translation products functioned as weak mitogens for mammary epithelial cells.

Fibrosarcoma of the transverse colon.

Colonic fibrosarcoma is extremely rare. This is a report of a patient with fibrosarcoma of the transverse colon, manifesting as an obstructive lesion with local perforation and abscess formation.