Heparin-binding (fibroblast) growth factors are potential autocrine regulators of esophageal epithelial cell proliferation

Summary A serum-free culture system supplemented with neural tissue extract for normal and tumor human esophagi was applied to the culture of mouse esophageal epithelium. Similar to mouse mesenchyme and skin epithelium, esophageal epithelial lines (MEE) emerged after serial culture. The cells had an apparent unlimited life span but retained morphology and other characteristics of normal epithelial cells. The cells formed a small cyst consisting of keratined squamous epithelium in syngenic hosts. A screen for growth factors that stimulated growth of the nonmalignant MEE cells in the absence of neural extract revealed that epidermal growth factor (EGF) and heparin-binding (fibroblast) growth factors (HBGF) were most effective. An HBGF-like activity was apparent in extracts of rapidly proliferating but not quiescent MEE cells at low or confluent densities. A cloned cell line (MEE/C8) was selected from MEE cell cultures in the absence of neural extract. MEE/C8 cells proliferated independent of either EGF or HBGF at rates equal to MEE cells, cell extracts exhibited HBGF-like activity at all stages of proliferation, and the cells formed large invasive tumors in syngenic hosts. The HBGF-like activity present in extracts of tumorigenic MEE/C8 and proliferating nonmalignant MEE cells had properties similar to HBGF-1 (acidic fibroblast growth factor). These results constitute a cultured mouse esophageal epithelial cell model for study of conversion of immortalized premalignant cells to malignant cells, and suggest that conversion from a state of cell cycle-dependent autocrine expression of one or more members of the HBGF family to a state of constitutive expression correlates with and may contribute to malignancy. The work was supported in part by grants CA37589 and DK35310 to Dr. McKeehan, from the National Cancer Institute, Bethesda, MD.     

The ATP synthase of Halobacterium salinarium (halobium) is an archaebacterial type as revealed from the amino acid sequences of its two major subunits.

The head piece of the A-type ATP synthase in an extremely halophilic archaebacterium, namely Halobacterium salinarium (halobium), is composed of two kinds of subunit, alpha and beta, and is associated with ATP-hydrolyzing activity. The genes encoding these subunits with hydrolytic activity have been cloned and sequenced. The putative amino acid sequences of the alpha and beta subunits deduced from the nucleotide sequences of the genomic DNA consist of 585 and 471 residues, respectively. The amino acid sequence of the alpha subunit of the halobacterial ATPase is 63 and 49% identical to the alpha subunits of ATPases from two other archaebacteria, Methanosarcina barkeri and Sulfolobus acidocaldarius, respectively. The sequence of the beta subunit is 66 and 55% identical to the beta subunits from these respective organisms. The homology between the alpha and beta subunits is around 30%. In contrast, the sequences of the halobacterial ATPase is less than 30% identical to F1 ATPase when any combination of subunits is considered. However, they are greater than 50% identical to a eukaryotic vacuolar ATPase when alpha and a, beta and b combinations are considered. These data fully confirm the first demonstration of this kind of relationship which was achieved by immunoblotting with an antibody raised against the halobacterial ATPase. We concluded that the archaebacterial ATP synthase is an A-type and not an F-type ATPase. This classification is also demonstrated by a "rooted" phylogenetic tree where halobacteria locate close to other archaebacteria and eukaryotes and distant from eubacteria.     

An endothelin receptor (ETA) antagonist isolated from Streptomyces misakiensis.

A competitive endothelin (ET) antagonist, BE-18257B, was isolated from the fermentation products of Streptomyces misakiensis. It is a novel cyclic pentapeptide, cyclo(-D-Glu-L-Ala-allo-D-Ile-L-Leu-D-Trp-), and binds to ETA receptors (ET-1 selective) in cardiovascular tissues, but not to ETB receptors (equally sensitive to isopeptides of ET family) in kidney, adrenal gland and cerebellum tissues. BE-18257B also antagonizes ET-1-induced vasoconstriction in rabbit iliac artery and pressor action in rats. Thus it is a selective ETA antagonist and should provide a valuable tool for elucidation of the pharmacological and pathophysiological roles of ET-1.     

The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein

We have purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimer's disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, respectively. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely associated with neuronal perikarya and dendrites. In the AD cortex, the number of GIF-positive astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.     

A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

The synthesis of evernitrose and 3-epi-evernitrose

Evernitrose (2,3,6-trideoxy-3-C-methyl-4-O-methyl-3-nitro-L-arabino-hexopyranose) was synthesized from methyl 2,6-dideoxy-4-O-methyl-α-L-erythro-hexopyranosid-3-ulose (2) through introduction of an amino group attached to the tertiary branching carbon by the method of Bourgeois, and subsequent oxidation of the amino group by m-chloroperoxybenzoic acid to a nitro group. 3-Cyano-3-O-mesylation of 2 by Bourgeois's method gave exclusively the desired product having the L-ribo configuration; furthermore, the β anomer of 2 gave the L-ribo and L-arabino products in the ratio of 1:2. The latter compound was converted into 3-epi-evernitrose by a similar sequence of reactions.     

The presence of an adenosine-5'-triphosphatase dependent on 6S tubulin and calcium ions in rat brain microtubules.

An ATPase activity was found in rat brain microtubules prepared by successive cycles of polymerization and depolymerization. On phosphocellulose column chromatography, the ATPase activity was recovered in the fraction eluted with 0.6 M KCl and containing the microtubule associated proteins. The ATPase activity was markedly stimulated by the addition of purified brain 6S tubulin, and the stimulation was dependent on the presence of Ca2+ ions. Approximately 50 pmol of purified 6S tubulin was required for the maximal stimulation in the presence of 8 microgram of microtubule associated proteins. The specific activity was 8 to 13 nmol of ATP hydrolyzed per min per mg of protein at 37 degrees C, and the Km value for ATP was 3 X 10(-5) M in the presence of added tubulin.