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Murine alpha-fetoprotein was synthesized in a wheat germ cell-free system in the presence of radioactive amino acids under the direction of alpha-fetoprotein messenger RNA isolated from mouse yolk sacs. The radiolabeled alpha-fetoprotein was isolated by immunoabsorption, and the amino acid residues at the NH2 terminus were determined by radioactive sequencing techniques. In a comparison to the NH2-terminal sequence of circulating alpha-fetoprotein, the in vitro-synthesized alpha-fetoprotein was found to contain an extra peptide 20 amino acids long linked at the NH2 terminus, the sequence of which is: (formula: see text). The molecular size, the hydrophobic nature, and the other properties of the peptide are consistent with the "leader" or "signal" piece found in the precursors of many other secretory proteins. This suggests that alpha-fetoprotein, the synthesis of which is limited primarily to fetal development, is produced in the form of the precursor as are secretory proteins in the adult tissues.

cDNA clones coding for a T-cell receptor beta-chain were isolated from a beef insulin/IAd-reactive T-cell hybridoma, A20.2.15, and its complete amino acid sequence was deduced. This beta-chain gene utilizes the same V beta segment as a thymocyte beta-chain gene (86T1) and rearranges to the 5' proximal J-C locus (J beta 1-C beta 1), thus providing definitive evidence of a beta-chain gene from a functional hybridoma that utilizes C beta 1. The amino acid sequence of the V beta gene in A20.2.15 is identical to 86T1, thus suggesting the absence of somatic mutation in the beta-chain of A20.2.15. Southern blot analysis revealed a somatic DNA rearrangement unique to the A20.2.15 hybridoma. The expression of this gene in the hybridoma was confirmed by RNA dot hybridization. All 24 beta-chain clones so far isolated from the A20.2.15 hybridoma contained C beta 1, suggesting that the beta-chain gene of the fusion partner BW5147 is not expressed in the hybridoma.

We describe experiments showing that the 5'-flanking region of the human alpha-fetoprotein (AFP) gene contains transcription control elements with characteristics of enhancers. The enhancer activity was detected and characterized by the ability to direct the expression of a linked chloramphenicol acetyltransferase (CAT) gene in transfected AFP-producing hepatoma cells in culture. The enhancer activity is cell-specific in that it occurs in hepatoma cells producing AFP, but not in non-AFP-producing hepatoma or nonhepatic cells. The active elements can direct CAT expression in conjunction with the SV40 promoter in an orientation- and position-independent manner. The sequences important for enhancer activity reside in the 400-base pair region between 3.3 and 3.7 kilobase pairs upstream of the AFP gene. This and proximal upstream regions contain multiple enhancer "core"-like sequences and other stretches of potential biological significance.

Zajdela rat ascites hepatoma cells have been reported to be 'nonsecretors' of alpha-fetoprotein (AFP). Because of the potential of such cells in the study of defective secretory mechanisms of AFP and other serum proteins, we concluded a detailed investigation of the production of AFP in Zajdela cells growing in vivo. Three approaches were used: radioimmunoassay of intracellular and extracellular AFP and assays of AFP mRNA by molecular hybridization and cell-free translation. Radioimmunoassay showed that the AFP concentration in the hepatoma cells was extremely low (1.2 micrograms/g) as compared to known AFP-producing tissues such as fetal liver (600 micrograms/g). The assays of AFP mRNA by hybridization and translation supported the low level of AFP production in Zajdela cells, and we consider such cells to be poor producers of AFP and therefore not suitable for the study of defective mechanisms for AFP secretion. The Zajdela tumor cell intracellular concentration of acute-phase alpha 2-macroglobulin (AP alpha 2M) measured by radioimmunoassay was also found to be low (3.0 micrograms/g) compared to liver of Zajdela-bearing rats (80 micrograms/g). In view of these low levels of synthesis of AFP and AP alpha 2M in Zajdela hepatoma cells, little biological significance may be attached to the apparent nonsecretion of these onco-fetal proteins.

Using a titration procedure, we measured the proportion of alpha-fetoprotein (AFP) and albumin mRNA in normal, regenerating, and preneoplastic rat livers. AFP mRNA constitutes approximately 0.006% of the polysomal polyadenylated RNA of normal livers and this proportion increases only slightly before the onset of DNA synthesis in liver regeneration induced by partial hepatectomy or CCl4 injury. In either model of liver regeneration, the proportion of AFP mRNA in polysomal RNA is highest approximately 24 h after the peak of DNA synthesis. The increase in the proportion of AFP mRNA in polysomal RNA is relatively small during liver regeneration (2-4-fold) but is larger (30-50-fold) in preneoplastic livers of rats fed a choline-deficient diet containing 0.1% ethionine. In contrast to those changes in AFP mRNA, albumin mRNA levels remain unchanged during liver regeneration and double in preneoplastic livers. Our results indicate that the concept of "retrodifferentiation" as it applies to liver regeneration and certain types of hepatic neoplasia needs reevaluation.

Tetrocarcin A, isolated from a Micromonospora culture showed activity against experimental i.p. inoculated tumors such as Ehrlich carcinoma, MH134 hepatoma, B16 melanoma. But it was not active against solid tumors such as sarcoma 180 and Ehrlich carcinoma. It was marginally active against the growth of solid Lewis lung carcinoma without prolonging the life span of the tumor-bearing mice. It was active against P388 leukemia (i.v.-i.v. system). It did not show myelosuppression and nephrotoxicity in mice. DNA and protein synthesis of P388 cells in culture were more significantly suppressed than RNA synthesis by tetrocarcin A.

Intracellular distribution of active mRNAs for alpha-fetoprotein (alphaFP) and albumin in fetal and adult mouse liver was studied. Livers were fractionated into nucleus and cytoplasm. The latter was further fractionated by sucrose density-gradient centrifugation into four subfractions, the top fraction containing soluble components, the 1.0 M sucrose layer containing primarily 80S ribosomes, the 1.5 M sucrose layer containing light polysomes and the pellets containing heavy polysomes. RNA was extracted from each fraction and its ability to direct the synthesis of alphaFP and albumin was determined in a mouse sarcoma 180 cell-free system. Distribution of alphaFP and albumin mRNAs in fetal mouse liver was similar; 2% in the nucleus, 98% in the cytoplasm, of which more than 90% was found in the polysome fractions. The results suggest that alphaFP and albumin mRNAs, once formed, are quickly and efficiently utilized for protein synthesis. The major proportion of albumin mRNA in adult mouse liver was also found to be associated with polysomes. However, the amount of translatable alphaFP mRNA was low in all subcellular fractions examined, suggesting that transcription or processing of alphaFP mRNA is defective in adult mouse liver.

In the absence of any specific literature on the isolation of RNA from mast cells, our initial attempts established that unusual measures would be needed to prepare acceptable yields of high quality RNA from peritoneal mast cells of normal adult rats. Accordingly, we developed procedures for the isolation and characterization of RNA from rat peritoneal mast cells (PMC) and basophilic leukemia cells (RBL). The significant components of the procedures include: separation and removal of mast cell granules to minimize contamination of RNA with proteins and proteoglycans; use of bentonite in phenol extractions; and repetition of extractions and precipitation. The amounts of total RNA extracted from PMC were about 15% of those from RBL, although the percentage mRNA of total RNA in PMC and RBL was similar (1.8 and 2.0%). Ribosomal RNA banding patterns in agarose gel electrophoresis and in vitro translation experiments indicate that the isolated RNA can be employed for analysis of molecular mechanisms of mast cell function and heterogeneity.

The potent protein kinase C inhibitors staurosporine, H-7, and UCN-01 were investigated for their effects on 12-0-tetradecanoylphorbol-13-acetate (TPA)--mediated CD4 down-regulation and on the augmentation of human immunodeficiency virus (HIV) expression. Staurosporine was the most effective TPA inhibitor for both of these actions. Because of its high cytotoxicity, the effect of H-7 on augmentation of HIV expression could not be determined. UCN-01 had no cytotoxic effect, but caused only little inhibition of the augmentation of HIV expression.

Mitochondrial particles were isolated from tobacco cells in tissue cultures which were disintegrated with pectinase. The pectinase treatment of the tissue cultures reduced the dry weight of the particulate preparations and eliminated glutinous material, but caused little change in oxidative and phosphorylative activities. The pectinase apparently did not affect the enzymatically active sites of the particles.