Ultrastructural characterization of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death in embryonic dopaminergic neurons.

Developing neuronal populations undergo significant attrition by natural cell death. Dopaminergic neurons in the substantia nigra pars compacta undergo apoptosis during synaptogenesis. Following this time window, destruction of the anatomic target of dopaminergic neurons results in dopaminergic cell death but the morphology is no longer apoptotic. We describe ultrastructural changes that appear unique to dying embryonic dopaminergic neurons. In primary cultures of mesencephalon, death of dopaminergic neurons is triggered by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and differs ultrastructurally from both neuronal apoptosis or typical excitotoxicity. AMPA causes morphological changes selectively in dopaminergic neurons, without affecting other neurons in the same culture dishes. Two hours after the onset of treatment swelling of Golgi complexes is apparent. At 3 h, dopaminergic neurons display loss of membrane asymmetry (coinciding with commitment to die), as well as nuclear membrane invagination, irregular aggregation of chromatin, and mitochondrial swelling. Nuclear changes continue to worsen until loss of cytoplasmic structures and cell death begins to occur after 12 h. These changes are different from those described in neurons undergoing either apoptosis or excitotoxic death, but are similar to ultrastructural changes observed in spontaneous death of dopaminergic neurons in the natural mutant weaver mouse.

Mechanism of PAP I gene induction during hepatocarcinogenesis: clinical implications.

Pancreatitis-associated protein I (PAP I) is a secretory protein first described as an acute phase reactant during acute pancreatitis. Recently, induction of the PAP I gene was also described in liver during hepatocarcinogenesis. To investigate the molecular mechanisms of this induction, we used constructs carrying progressive deletions of the PAP I promoter fused to the CAT gene. We showed that the silencer conferring tissue specificity on the PAP I gene was inactive in hepatoma cells. Then, in an vitro transcription system, we compared the transcription capacity of nuclear extracts from normal liver and HepG2 cells on constructs containing the silencer. The results confirmed that a trans-acting factor interacting with the PAP I silencer was present in liver cells and absent from hepatoma cells. On the other hand, immunohistochemistry showed that PAP I was expressed in a limited number of transformed hepatocytes. It was concluded that expression of PAP I in hepatocarcinoma occurred through inactivation of its silencer element and was not concomitant in all malignant cells. On that basis, we assayed PAP I in serum from patients with chronic hepatitis, liver cirrhosis or hepatocarcinoma. PAP I levels were normal in chronic active or persistent hepatitis, significantly higher in cirrhosis and strongly elevated in hepatocarcinoma. Because those clinical entities often develop in that sequence, serum PAP I appeared as a potential marker of hepatocarcinoma development.

Immunocytochemical localization of pancreatitis-associated protein in human small intestine.

Pancreatitis-associated protein (PAP) is a lectin-related protein barely detectable in normal pancreas but overexpressed by this tissue during the acute phase of the pancreatitis. We describe in this report that PAP is constitutively expressed in the human intestinal tract. Northern blot analysis with pancreatic cDNA as probe shows the presence of a transcript in the jejunum that has the same electrophoretic mobility as the pancreatic mRNA. No signal was detected in colon, however. In addition, immunoblotting assays, utilizing specific rabbit immunosera prepared against PAP, revealed the presence of a protein of 16,000 Da (as in pancreatic juice) in the homogenate of jejunum, but not of the colon. When the same antibodies were used for tissule localization of the protein, positive immunoreactivity was observed on Paneth cells and in some goblet cells located in jejunum at the bottom of the crypts. No staining was observed in colon.

Human pancreatitis-associated protein. Messenger RNA cloning and expression in pancreatic diseases.

A human pancreatic cDNA library was screened with the cDNA encoding rat "pancreatitis-associated protein" (PAP). The selected clone encoded a secretory protein structurally related to rat PAP. The protein had the same size as rat PAP and showed 71% amino acid identity, the six half-cystines being in identical positions. Domains of the proteins showing homologies with calcium-dependent lectins were also conserved. In addition, expression in pancreas of the genes encoding the human protein and rat PAP showed similar characteristics: both were expressed at very low levels in control tissue and overexpressed during the acute phase of pancreatitis, contrary to most secretory products. The human protein was therefore named human pancreatitis-associated protein (PAP-H). Antibodies raised to a synthetic peptide of PAP-H detected a single band with an M(r) compatible with PAP-H in Western blot analysis of proteins extracted from a pancreas presenting with acute pancreatitis. In that tissue, the protein could be immunolocalized to the apical regions of acinar cells. An immunoassay was also constructed to quantify the protein in serum. Elevated PAP-H levels were observed in patients with acute pancreatitis and in some patients with chronic pancreatitis. Values were close to background in healthy subjects and in patients with other abdominal diseases. These results confirm that PAP-H synthesis increases during inflammation and suggest a possible use of the protein as biological marker of acute pancreatitis.