Two cDNAs were cloned that encode proteins (Pan-1 and Pan-2, with predicted molecular masses of 67.4 and 67.7 kD, respectively} that bind to a conserved transcriptional regulatory element present in pancreatic exocrine genes. The cDNAs are virtually identical in the region that encodes the amino-terminal 525 amino acids; they differ only by a 3-nucleotide insertion that results in the addition of a single amino acid. The cDNAs, however, code for related, but divergent, carboxy-terminal regions. Expression of the cloned cDNAs in HeLa cells activates expression of a cotransfected chimeric gene containing multimers of the pancreatic conserved element linked to a minimal promoter. Pan-1 and Pan-2 bind to the consensus sequence CAc/cCTGT/o the CACCTGTC motif, which is present in transcriptional control elements of several genes. Both Pan-1 and Pan-2 bind to related motifs present in the rat insulin enhancer as well as a DNA segment containing the SV40 AP-4 element. The Pan-1 and Pan-2 cDNAs display extensive sequence identity with partial cDNA clones encoding El2 and E47, which bind to the immunoglobulin KE2 c/s-active element, and may be derived from the same gene. These factors belong to a set of genetically distinct molecules, including AP-4 and MLTF, that bind to the CACCTGTC motif or related sequences. The Pan/E12,E47 proteins also show structural similarity with the Drosophila daughterless protein, MyoD, Myogenin, and Myf-5.

Polypeptide growth factors, including epidermal growth factor (EGF), play a central role in regulating hepatocyte growth both in vivo and in primary culture. To characterize EGF gene expression in the pathogenesis of regenerative cirrhotic fibrosis, we employed biotinylated antisense oligonucleotide probes to localize hepatic mRNA transcripts in situ. In control tissue and regenerative hepatic nodules, EGF receptor (EGFR) mRNA transcripts were expressed constitutively. In contrast, oligonucleotide probes targeting the human EGF coding region showed that EGF transcription was extremely low in control liver but was highly elevated and localized to regenerative hepatic nodules and bile duct epithelia of cirrhotic liver. To determine whether EGF mRNA accumulation accompanied a comparable increase in the EGF peptide, we performed immunohistochemistry using an antibody specific for the nonprocessed peptide aminoterminus. We observed that positive localized EGF staining paralleled its mRNA transcript. These results indicate that EGF upregulation is a characteristic of cirrhotic liver disease and suggest that persistent de novo ligand synthesis and its signaling contribute to an autocrine-mediated hepatocyte proliferation within the regenerative nodule.

Upon fertilization, sea urchin eggs (Stronglyocentrotus pupuratus) release a protease into the surrounding sea water. This protease is in a particulate form which can be solubilized. The soluble form was purified by affinity chromatography on columns of immobilized soybean trypsin inhibitor. The purified enzyme is similar to bovine trypsin both in molecular weight (22500) and in susceptibility to inhibitors such as diisopropyl phosphofluoridate and soybean trypsin inhibitor. In contrast, extracts of unfertilized eggs appear to contain an inactive form of the enzyme which can be activated by dialysis at pH 4.6. The enzyme, as purified from extracts activated in this manner, was similar in its properties to that from fertilized eggs.

We have found that chicken reticulocytes respond to elevated temperatures by the induction of only one heat shock protein, HSP70, whereas lymphocytes induce the synthesis of all four heat shock proteins (89,000 mol wt, HSP89; 70,000 tool wt, HSP70; 23,000 mol wt, HSP23; and 22,000 mol wt, HSP22). The synthesis of HSP70 in lymphocytes was rapidly induced by small increases in temperature (2°-3°C) and blocked by preincubation with actinomycin D. Proteins normally translated at control temperatures in reticulocytes or lymphocytes were not efficiently translated after incubation at elevated temperatures. The preferential translation of mRNAs that encode the heat shock proteins paralleled a block in the translation of other cellular proteins. This effect was most prominently observed in reticulocytes where heat shock almost completely repressed c~-and/3-globin synthesis.HSP70 is one of the major nonglobin proteins in chicken reticulocytes, present in the nonheat-shocked cell at approximately 3 x 106 molecules per cell. We compared HSP70 from normal and heat-shocked reticulocytes by two-dimensional gel electrophoresis and by digestion with Staphylococcus aureus V8 protease and found no detectable differences to suggest that the P70 in the normal cell is different from the heat shock-induced protein, HSP70. P70 separated by isoelectric focusing gel electrophoresis into two major protein spots, an acidic P70A (apparent pl = 5.95) and a basic P70B (apparent pl = 6.2). We observed a tissue-specific expression of P70A and P70B in lymphocytes and reticulocytes. In lymphocytes, P70A is the major 70,000-mol-wt protein synthesized at normal temperatures whereas only P70B is synthesized at normal temperatures in reticulocytes. Following incubation at elevated temperatures, the synthesis of both HSP70A and HSP70B was rapidly induced in lymphocytes, but synthesis of only HSP70B was induced in reticulocytes.Procaryotic and eucaryotic cells respond to elevated temperatures by the synthesis of the ubiquitous "heat shock" proteins, a family of proteins of 80-90,000 mol wt, 70,000 mol wt, and smaller proteins of 20-30,000 mol wt (36). Other cellular effects of heat shock include altered cell morphology (37, 40), inhibition of DNA synthesis affecting the cell cycle and mitosis (12, 34), a generalized repression in RNA polymerase ll-dependent transcription (10, 38), and the selective translation of heat shock messenger RNAs (23,25). Heat shock proteins are also induced in various cells after exposure to diverse and apparently unrelated chemicals including heavy metals (Cd, Cu, Zn), inhibitors of energy metabolism (arsenite), amino acid analogues (canavanine, 5-methyltryptophan), chelators (disulfiram), and infection with certain viruses such as adenovirus 5 (2, 15, 17, 21, 32).Among diverse organisms as yeast, Drosophila, chicken, mouse, and human, these induced proteins are highly conserved. Kelley et al. (18) have shown that heat shock proteins from different eucaryotes are antigenically related to the chicken heat shock pro...