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.     

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.     

Recovery of maize seedling growth, development and photosynthetic efficiency after initial growth at low temperature

In order to investigate the mechanisms of maize adaptation to temperate climate, we studied photosynthetic efficiency, as evaluated by means of phiPSII and chloroplast ultrastructure, as well as growth and development of two inbred lines (the chilling-tolerant KW 1074 and the chilling-sensitive CM 109) under laboratory conditions. Plants were grown from seed to the 3rd leaf stage at a suboptimal temperature (14 degrees C/ 12 degrees C) and then the temperature was increased to 24 degrees C/22 degrees C. To verify the results obtained with the two model lines, twelve inbred lines were tested under both laboratory and field conditions. Initial growth at low temperature affected the chloroplast ultrastructure and photosynthetic efficiency, and this was more pronounced in CM 109 than in KW 1074 plants. The differences between the two lines were particularly pronounced in leaf 5. One week after the onset of favourable conditions, mesophyll chloroplast grana in the CM 109 line were small and thylakoids were developed only poorly. Also, thylakoids in bundle sheath chloroplasts were less frequent in CM 109 than in KW 1074. However, two weeks after the temperature increase, the ultrastructure of chloroplasts of the 5th leaf no longer differed distinctly between the two lines. One should note that in both lines, only the 7th and younger leaves reached a chloroplast ultrastructure and phiPSII indistinguishable from those of control plants. In general, the recovery of photosynthetic efficiency followed the development of leaves. It was delayed in the CM 109 more than in the KW 1074 inbred line relative to control plants grown continuously at the optimal temperature. The growth difference of 2-3 days between the two lines persisted even after the growth temperature was elevated. This suggested that the primary factor responsible for the different chilling-sensitivities of the two model lines was leaf development and the differences in development of the photosynthetic apparatus had only a secondary role. The delay in leaf development appeared as early as the stage of the 1st leaf. The same delay was observed when only the shoot apex was cooled. The importance for further recovery of the early stages of morphogenesis was confirmed by a correlation of Laboratory and field data that were obtained using a set of 12 inbred lines. Our results suggest that early stages of shoot morphogenesis determine the duration of the vegetative phase in cool regions, since the delay in growth at a low temperature cannot be compensated for during later growth at a higher temperature.     

Expression of alpha-synuclein in different brain parts of adult and aged rats.

The synucleins are a family of presynaptic proteins that are abundant in neurons and include alpha-, beta, and gamma-synuclein. Alpha-synuclein (ASN) is involved in several neurodegenerative age-related disorders but its relevance in physiological aging is unknown. In the present study we investigated the expression of ASN mRNA and protein in the different brain parts of the adult (4-month-old) and aged (24-month-old) rats by using RT-PCR technique and Western blot, respectively. Our results indicated that mRNA expression and immunoreactivity of ASN is similar in brain cortex, hippocampus and striatum but markedly lower in cerebellum comparing to the other brain parts. Aging lowers ASN mRNA expression in striatum and cerebellum by about 40%. The immunoreactivity of ASN in synaptic plasma membranes (SPM) from aged brain cortex, hippocampus and cerebellum is significantly lower comparing to adult by 39%, 24% and 65%, respectively. Beta-synuclein (BSN) was not changed in aged brain comparing to adult. Age-related alteration of ASN may affect the nerve terminals structure and function.