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.     

Epistasis and the Genetic Divergence of Photoperiodism between Populations of the Pitcher-Plant Mosquito, Wyeomyia Smithii

Parallel crosses between each of two southern (ancestral) and one northern (derived) population of the pitcher-plant mosquito, Wyeomyia smithii, were made to determine the genetic components of population divergence in critical photoperiod, a phenological trait that measures adaptation to seasonality along a climatic gradient. Joint scaling tests were used to analyze means and variances of first- and second-generation hybrids in order to determine whether nonadditive genetic variance, especially epistatic variance, contributed to divergence in critical photoperiod. In both crosses, digenic epistatic effects were highly significant, indicating that genetic divergence cannot have resulted solely from differences in additively acting loci. For one cross that could be tested directly for such effects, higher order epistasis and/or linkage did not contribute to the divergence of critical photoperiod between the constituent populations.     

A mutant ofDunaliella parva CCAP 19/9 leaking large amounts of glycerol into the medium

A mutant of the halotolerant green algaDunaliella parva, which leaks large amounts of intracellular glycerol into the surrounding medium, was isolated. The mutant has potential applications in the commercial production of glycerol on a large scale since there is no need to extract glycerol from the cells. The mutant was compared with the wild type and it was found that, despite the leakage of glycerol, the mutant showed the same growth rate as the wild type. However, when the rates of oxygen evolution and uptake and intracellular starch content between mutant and wild type were compared at high salinity, considerable differences were found.     

Quantitation of the T cell deficiency in RFM mice with host versus graft disease.

Host versus graft (HVG) syndrome is the fatal complex of lesions which has been observed in six inbred strains of mice following the perinatal inoculation of related F₁ hybrid spleen cells. Morphological studies have indicated that the key lesion is the depletion of peripheral T lymphocytes due to inflammatory destruction and failure of the thymus to replace them. In the present studies, tests of T-cell function were done on RFM mice, which had developed HVG disease following perinatal inoculations of (T₆ × RFM)F₁ spleen cells. As compared to control values, HVG spleen cell suspensions showed loss of reactivity to phytohemagglutinin (PHA) = 90%, to concanavalin A (Con A) = 94%, to (T₆ × RFM)F, cells in the mixed lymphocyte reaction (MLR) = 82%, to DBA cells in MLR = 94%, and to DBA mastocytoma cells in cell-mediated lympholysis (CML) = 95%. Lymph node cell suspensions showed losses of reactivity to PHA = 83%, to Con A = 62%, to (T₆ × RFM)F₁ cells in the MLR = 91%, and to DBA cells in the MLR = 77%. The CML activity of nodal cells to DBA mastocytoma cells varied widely from 12 to 273% of control values, and averaged 121%. Filtration of HVG spleen cells through nylon fiber columns failed to restore low responses to PHA to normal values. This suggested that the macrophage-like, adherent accessory cells were not acting as suppressors of T-cell responses in HVG disease. The deficits in all T-cell-mediated functions tested so far, appeared to correlate very well with quantitative morphological studies which showed the loss of 98% of the small lymphocytes normally present in the thymic dependent portions of the splenic white pulp. It is suggested that experimental HVG disease may serve as a model for immunodeficiency syndromes of the Nezelof type which are also characterized by T-cell deficiency, poor primary antibody responses, and the presence of variable amounts of serum immunoglobulins.     

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.