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To test the hypothesis that ras activation is involved in the final stages of breast cancer progression, we analyzed tumor DNA derived from 60 different patients and extracted from 40 invasive primary breast tumors, seven lymph node and skin metastases, nine metastatic effusions, and five established breast cancer cell lines. The polymerase chain reaction technique was used to amplify DNA fragments containing Kirsten-(Ki-), Harvey-(Ha-), and N-ras codons 12, 13, and 61 which were then probed on slot-blots with labeled synthetic oligomers to detect nonconservative single base mutations. Activating mutations were found in one of 40 primary tumors (Ki-ras codon 13), zero of seven lymph node and skin metastases, one of nine metastatic effusions (Ki-ras codon 12), and two of five cell lines (Ki-ras codons 12 and 13). These results indicate that activating ras mutations are rarely involved in either the initiation or metastatic progression of human breast cancer.

A neutral proteinase, located on the surface of human granulocytes and lymphocytes, degraded bovine parathyroid hormone in vitro. The observed sites of cleavage were between residues 5 and 6 (--ile--gln--), 8 and 9 (--met--his--), 38 and 39 (--gly--ala--) and 41 and 42 (--ile--ala--). Possible physiological implications of these findings are discussed.

The ocean pout (Macrozoarces americanus) produces a set of antifreeze proteins that depresses the freezing point of its blood by binding to, and inhibiting the growth of, ice crystals. The amino acid sequences of all the major components of the ocean pout antifreeze proteins, including the immunologically distinct QAE component, have been derived by Edman degradation. In addition, sequences of several minor components were deduced from DNA sequencing of cDNA and genomic clones. Fifty percent of the amino acids are perfectly conserved in all these proteins as well as in two homologous sequences from the distantly related wolffish. Several of the conserved residues are threonines and asparagines, amino acids that have been implicated in ice binding in the structurally unrelated antifreeze protein of the righteye flounders. Aside from minor differences in post-translational modifications, heterogeneity in antifreeze protein components stems from amino acid differences encoded by multiple genes. Based on genomic Southern blots and library cloning statistics there are 150 copies of the 0.7-kilobase-long antifreeze protein gene in the Newfoundland ocean pout, the majority of which are closely linked but irregularly spaced. A more southerly population of ocean pout from New Brunswick in which the circulating antifreeze protein levels are considerably lower has approximately one-quater as many antifreeze protein genes. Thus, there appears to be a correlation between gene dosage and antifreeze protein levels, and hence the ability to survive in ice-laden seawater. Southern blot comparison of the two populations indicates that the differences in gene dosage were not generated by a simple set of deletions/duplications. They are more likely to be the result of differential amplification.

A modification of the polymerase chain reaction technique (PCR) technique, a primer-mediated enzymatic amplification of specific target sequences in genomic DNA, was used to introduce point mutations into copies of human ras oncogene sequences, and to amplify these mutated copies approximately 10(6)-fold. Of the two flanking oligomers used to amplify the DNA, one contained a single base mismatch with the targeted gene segment, either codon 12 or 61 from the Kirsten ras oncogene. Double-stranded fragments harboring any point mutation can be generated using the appropriate oligomer and constitute greater than 99.999% of the PCR-amplified fragments. The amplified DNA fragments are readily slot-blotted to nylon membranes to serve as positive hybridization controls in the search for gene mutations in human tissue specimens. The synthesis of single base mismatched DNA fragments was also used to demonstrate that oncogene mutations can be detected from mixed DNA populations as might be present in primary tumor specimens, even when the mutation of interest is present in only 5% of the amplified sample DNA.

The primary translation product encoded by sea raven antifreeze protein mRNA was labeled during cell-free synthesis with [3H]leucine. N-terminal sequencing of the immunoselected translation product showed that the third AUG in the mRNA is used as the initiating methionine codon. The antifreeze protein precursor is therefore 163 amino acids long. Amino acid analysis and sequencing of the deblocked N-terminal peptide from the mature circulating form of the antifreeze indicated that glutamine at position 35 is the N-terminal residue. The most likely site of signal peptide cleavage is after alanine at position 17, suggesting that the sea raven antifreeze protein is produced as a preproprotein. Analysis of slot blots indicates that the gene for the antifreeze protein is present in 12-15 copies in the sea raven genome. A representative gene copy was sequenced. It is split into six exons spanning 2.2 kilobase pairs and, based on composite maps of genomic clones, is not accompanied by a second copy within at least 25 kilobase pairs of flanking DNA. The transcription start site was determined by primer extension. Ninety base pairs upstream from this point, beyond the CAAT and TATA boxes, is a putative cis-acting regulatory element in the form of a triplicated 21-base pair tandem repeat.

Previous experiments have shown that cultured human fibroblasts possess a cell-surface proteinase (the growth-related proteinase; GRP) which is essential to cell proliferation. In the present work, proteinase inhibition in defined and complex serum-free media and in pre-conditioned normal medium, still resulted in a corresponding inhibition of cell proliferation. Proteinase inhibition also blocked the action of a range of peptide growth factors and of a phorbol ester. Elevated extracellular calcium concentrations were still mitogenic in the presence of proteinase inhibitors. Proteinase inhibition did not affect the mobilisation of intracellular calcium, nor the metabolism of inositol phosphate derivatives in response to a mitogenic stimulus.

The serum killing of Escherichia coli ML308 225 and PB94 was inhibited by lipopolysaccharide extracted from either organism, but not by lipopolysaccharide from three pathogenic Enterobacteriaceae.

Short-term cultures of normal human mammary epithelial cells were used to determine the extent to which c-myc, c-Ha-ras1, and c-erbB-2 proto-oncogenes were expressed in proliferating normal cells. This level of expression was compared with that of primary tumor cells, malignant effusion cells, or permanently established breast cancer cell lines. Pure preparations of epithelial organoids from seven different reduction mammoplasty tissue samples yielded proliferating normal epithelial cells upon short-term tissue culture. In every sample, proto-oncogene transcript levels increased upon short-term culture of the epithelial cells. These levels often exceeded by 10-fold the levels measured in uncultured organoids from the same tissue. In four of the seven cultured normal breast samples, at least one of the proto-oncogenes increased its expression to a level equaling or exceeding that found in a proliferating breast cancer cell line, MCF7. One effusion metastasis sample and two primary ductal adenocarcinomas were also examined for proto-oncogene expression. The effusion metastasis sample expressed high levels of c-erbB-2 messenger RNA, in accord with its amplified gene copy number; otherwise, the levels of proto-oncogene transcripts were low in unprocessed tumor and uncultured organoids, but they increased with proliferation of the tumor cells in culture. These results indicate that the variable expression of these proto-oncogenes observed in breast biopsy specimens needs to be controlled for cellular growth rate or proliferation index. Furthermore, these findings suggest that dysregulated proto-oncogene expression, rather than overexpression per se, needs to be evaluated as a possible mechanism contributing to the development of human breast cancer.

Specific antibodies and protein proteinase inhibitors will inhibit cell-surface proteinase activity on human fibroblasts and cause a concomitant inhibition of DNA synthesis and of cell multiplication. An insolubilized proteinase inhibitor also inhibits cell multiplication. The same reagents partially inhibit the multiplication of mouse L cells, both in monolayer and suspension culture, and inhibit the mitogenic effect of epidermal growth factor (EGF) on both types of cell.

The size-distribution of liver glycogen was shown to be distinctly affected by the anti-inflammatory drugs salicylate and indomethacin. By measurement of the incorporation of radioactive glucose into glycogen, salicylate was shown to have a depressing effect on overall liver glycogen metabolism. These effects appear to arise from the stabilizing of the lysosome by the drugs. The incorporation, via liposomes, of purified anti-1,4-alpha-glucosidase activity and in the content of high-molecular-weight glycogen. These changes are increased by prolonged liposomal antibody treatment and suggest that a possible feedback control mechanism operates in the incorporation of glycogen into lysosomes. These experiments may be useful as a model of glycogen turnover and its failure in glycogenosis type II (Pompe's disease).

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.

Isolated outer membranes and outer-membrane extracts from Escherichia coli ML308-225 in the early-exponential growth phase contain more protein than do corresponding preparations from late-exponential- or stationary-phase bacteria. Isotope-dilution experiments show that this is due to a loss of protein from the membrane during the exponential growth phase. Inhibition of bacterial growth and protein synthesis stabilizes the outer-membrane-protein concentration. Protein synthesis in the absence of bacterial growth results in higher concentrations of protein in the outer membrane.

1. Inhibition of a human fibroblast cell-surface proteinase with alpha-1-antitrypsin also inhibited cell proliferation, confirming earlier observations. 2. Although a similar proteinase inhibition could be observed with human tumour cells, the growth of these cells was unaffected. 3. Plasminogen activators on the cell surface and in culture media could be inhibited with specific antibodies, but there was no corresponding effect on the growth of any of the cell types tested.

The antifreeze protein genes of the wolffish (Anarhichas lupus) constitute a large multigene family of 80 to 85 copies, which can be classified into two sets. One-third of the genes were linked but irregularly spaced. The other two-thirds were organized as 8-kilobase-pair (kbp) tandem direct repeats that each contained two genes in inverted orientation; DNA sequence analysis suggests that both genes are functional. Except for a single region specific to each gene, the genes and their immediate flanking sequences were 99.2% identical. This degree of identity ended soon after a putative transcription termination sequence; as the 3' ends of the genes were only 1.3 kbp apart, these sequences might confer mutual protection from interference by transcriptional runoff. A Southern blot of wolffish DNA restricted with enzymes that do not cut within the tandem repeats indicated that the repeats were clustered in groups of six or more. The organization of antifreeze protein genes in the wolffish was very similar to that in the unrelated winter flounder, which produces a completely different antifreeze. This similarity might reflect common dynamics by which their progenitors adapted to life in ice-laden sea water.

Antibodies specific for a purified proteinase from the cell-surface of human leukocytes were used to prepare an antibody affinity chromatography column. This column bound a variety of proteins from extracts of human cells and tissues and from human body fluids, indicating that proteins immunologically related to the leukocyte proteinase are widespread in the human body. For human urine and a human fibroblast extract, a single protein species with serine proteinase activity was eluted from the column in each case. Small quantities of a heterogeneous protein fraction were also weakly bound to the column from an extract of mouse submaxillary glands.

The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10-12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.

We have used genomic Southern blots and restriction maps of genomic clones to examine the organization of the antifreeze protein multigene family in the winter flounder. The majority of the approximately equal to 40 antifreeze protein (AFP) genes in this fish are present in 7- to 8-kilobase-pair (kbp) elements of DNA, which are iterated as tandem direct repeats. Each repeat contains a single antifreeze protein gene that is 1 kbp long, and all of these genes have the same transcriptional orientation. Although the repeated elements are highly homologous, they do show some restriction site and restriction length polymorphisms. When flounder genomic DNA is digested with restriction endonucleases that do not cut within the repeats, most of the antifreeze protein genes reside in fragments that are at least 40 kbp long, representing clusters of five or more repeats in tandem. After genomic DNA is digested with Xba I or Xho I, these genes are present in fragments of exceptionally high molecular weight, suggesting that the clusters themselves are grouped together in the genome. The AFP gene locus may have evolved by gene amplification as recently as 10(6) years ago in response to the onset of the Cenozoic ice age.

Purified penicillinase, in gram quantities, has been prepared from Escherichia coli strain W3310 by using methods developed to handle large amounts of material. The final product had a specific enzyme activity of 3.08 units/mug of protein, which was over twice as high as that reported previously (Datta & Richmond, 1966). The purified enzyme was similar to that from E. coli strain TEM, but different in molecular weight and some other respects. The differences observed may be a result of the greater purity obtained.