Cultured endothelial cells produce heparinlike inhibitor of smooth muscle cell growth

Using cultured cells from bovine and rat aortas, we have examined the possibility that endothelial cells might regulate the growth of vascular smooth muscle cells. Conditioned medium from confluent bovine aortic endothelial cells inhibited the proliferation of growth-arrested smooth muscle cells. Conditioned medium from exponential endothelial cells, and from exponential or confluent smooth muscle cells and fibroblasts, did not inhibit smooth muscle cell growth. Conditioned medium from confluent endothelial cells did not inhibit the growth of endothelial cells or fibroblasts. In addition to the apparent specificity of both the producer and target cell, the inhibitory activity was heat stable and not affected by proteases. It was sensitive flavobacterium heparinase but not to hyaluronidase or chondroitin sulfate ABC lyase. It thus appears to be a heparinlike substance. Two other lines of evidence support this conclusion. First, a crude isolate of glycosaminoglycans (TCA-soluble, ethanol-precipitable material) from endothelial cell-conditioned medium reconstituted in 20 percent serum inhibited smooth muscle cell growth; glycosaminoglycans isolated from unconditioned medium (i.e., 0.4 percent serum) had no effect on smooth muscle cell growth. No inhibition was seen if the glycosaminoglycan preparation was treated with heparinase. Second, exogenous heparin, heparin sulfate, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate ABC, and hyaluronic acid were added to 20 percent serum and tested for their ability to inhibit smooth muscle cell growth. Heparin inhibited growth at concentrations as low as 10 ng/ml. Other glycosaminoglycans had no effect at doses up to 10 μg/ml. Anticoagulant and non- anticoagulant heparin were equally effective at inhibiting smooth muscle cell growth, as they were in vivo following endothelial injury (Clowes and Karnovsk. Nature (Lond.). 265:625-626, 1977; Guyton et al. Circ. Res. 46:625-634, 1980), and in vitro following exposure of smooth muscle cells to platelet extract (Hoover et al. Circ. Res. 47:578-583, 1980). We suggest that vascular endothelial cells may secrete a heparinlike substance in vivo which may regulate the growth of underlying smooth muscle cells.     

Ammonium Excretion by an l-Methionine-dl-Sulfoximine-Resistant Mutant of the Rice Field Cyanobacterium Anabaena siamensis

An ammonium-excreting mutant (SS1) of the rice field nitrogen-fixing cyanobacterium Anabaena siamensis was isolated after ethyl methanesulfonate mutagenesis by selection on 500 muM l-methionine-dl-sulfoximine. SS1 grew in the presence and absence of (l)-methionine-dl-sulfoximine at a rate comparable to that of the wild-type strain, with a doubling time of 5.6 h. The rate of ammonium release by SS1 depended on cell density; it peaked at the 12th hour of growth with 8.7 mumol mg of chlorophyll h (at a chlorophyll concentration of 5 mug ml) and slowed down to almost nil at the fourth day of growth. A similar pattern of release by immobilized SS1 was observed between 12 to 20 h after loading alginate beads in packed-bed reactors at the rate of 11.6 mumol mg of chlorophyll h. The rate was later reduced significantly due to the fast growth of SS1 on the substrate. Prolonged release of ammonium at the peak level was achieved only by maintaining SS1 under continuous cultivation at low chlorophyll levels (5 to 7 mug ml). Under these conditions, nitrogen fixation in the mutant was 30% higher than that in its parent and glutamine synthetase activity was less by 50%. Immunoblot analysis revealed that SS1 and its parent have similar quantities of glutamine synthetase protein under ammonium excretion conditions. In addition, a protein with a molecular weight of about 30,000 seems to have been lost, as seen by electrophoretic separation of total proteins from SS1.     

Subcellular localization of DNA-binding protein BA by immunofluorescence and immunoelectron microscopy

Nonhistone protein BA has been shown to decrease in amount in the chromatin of growth- stimulated normal rat liver (Yeoman et al. 1975. Cancer Res. 35:1249-1255) and in mitogen-stimulated normal human lymphocytes (Yeoman et al. 1976. Exp. Cell Res. 100:47- 55.). Subsequently, protein BA was purified and was shown to prefer to bind to double- stranded A-T-rich DNAs (Catino et al. 1978. Biochemistry. 17:983-987.). Immunization of rabbits with highly purified protein BA has resulted in the production of a specific antibody. A specific immunoreactivity for chromosomal protein BA has been demonstrated by immunoelectrophoresis and double antibody immunoprecipitation analysis with rabbit anti-BA immunoglobulin and IgG fractions. Light microscope examination of normal rat liver crysections by the indirect immunofluorescence procedure has demonstrated a cytoplasmic as well as a nuclear localization for protein BA with a pronounced perinucleolar fluorescence. Immunoelectron microscopy employing the peroxidase antiperoxidase method of antigen localization has confirmed the immunofluorescence data and has show a heterochromatin localization for protein BA. The relationship of the localization of protein BA to gene control in quiescent cells or to configurations of heterochromatin as well as the marked reduction in the amounts of protein BA which occur in stimulated growth states remains to be defined.     

Partial restoration of antibacterial activity of the protein encoded by a cryptic open reading frame (cyt1Ca) from Bacillus thuringiensis subsp. israelensis by site-directed mutagenesis

Insecticidal crystal proteins of Bacillus thuringiensis belong to two unrelated toxin families: receptor-specific Cry toxins against insects and Cyt toxins that lyse a broad range of cells, including bacteria, via direct binding to phospholipids. A new cyt-like open reading frame (cyt1Ca) encoding a 60-kDa protein, has recently been discovered (C. Berry et al., Appl. Environ. Microbiol. 68:5082-5095, 2002). Cyt1Ca displays the structure of a two-domain fusion protein: the N-terminal moiety resembles the full-length Cyt toxins, and the C-terminal moiety is similar to the receptor-binding domains of several ricin-like toxins, such as Mtx1. Neither the larvicidal activity of cyt1Ca expressed in Escherichia coli nor the hemolytic effect of His-tagged purified Cyt1Ca has been observed (R. Manasherob et al., unpublished). This was attributed to five amino acid differences between the sequences of its N-terminal moiety and Cyt1Aa. The 3' end of cyt1Ca was truncated (removing the ricin-binding domain of Cyt1Ca), and six single bases were appropriately changed by site-directed mutagenesis, sequentially replacing the non-charged amino acids by charged ones, according to Cyt1Aa, to form several versions. Expression of these mutated cyt1Ca versions caused loss of the colony-forming ability of the corresponding E. coli cells to different extents compared with the original gene. In some mutants this antibacterial effect was associated by significant distortion of cell morphology and in others by generation of multiple inclusion bodies spread along the cell envelope. The described deleterious effects of mutated cyt1Ca versions against E. coli may reflect an evolutionary relationship between Cyt1Aa and Cyt1Ca.     

Mosquito larvicidal activity of transgenic Anabaena strain PCC 7120 expressing combinations of genes from Bacillus thuringiensis subsp. israelensis.

Various combinations of the genes cryIVA (cry4A), cryIVD (cry11A), and p20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing cyanobacterium Anabaena sp. strain PCC 7120 by means of Escherichia coli-Anabaena shuttle vector pRL488p and were expressed under control of two tandem strong promoters, a cyanobacterial promoter (PpsbA) and an E. coli T7 promoter (PA1). Two of the clones carrying cryIVA plus cryIVD, one with p20 and one without p20, displayed toxicity against third-instar larvae of Aedes aegypti at levels greater than any level previously reported for transgenic cyanobacteria.