ENZYME ACTIVITIES ASSOCIATED WITH RIBOSOMES FROM SOYBEAN SEEDLINGS

Ribosomes from cotyledons of soybean seeds soaked overnightin water (resting-cotyledon ribosomes) and respective ribosomesfrom cotyledons (working-cotyledon ribosomes) and hypocotyls(hypocotyl ribosomes) of 5 day-old seedlings were prepared.These ribosomes mainly consisted of 80 S particles. However,hypocotyls contained 115 S particles in a small amount and working-cotyledonscontained 115 and 150 S particles which may correspond to thepolymers of ribosomes or polysomes. The abundant 80 S ribosomeswere fractionated by the sucrose density gradient centrifugation,and enzyme activities in the fractionated ribosomes were estimated.RNase, PDase, acid phosphatase, 5'-nucleotidase, XTPase, peroxidaseand ß-glucosidase were found in the ribosomes. Theenzyme activities were lower in the resting-cotyledon ribosomesand higher in the hypocotyl ribosomes. Working-cotyledon ribosomesshowed the middle of them. All these enzymes were also foundin the cytoplasmic solution (supernatant) of cotyledon and hypocotylcells. However, RNase, PDase, 5'-nucleotidase and peroxidasewere concentrated in ribosomes, and the specific activitiesof 5'-nucleotidase and ß-glucosidase were increasedby washing the ribosomes. The status of the enzymes found inthe ribosomes was discussed. (Received May 12, 1966; )     

THREE-DIMENSIONAL STRUCTURE OF EXTRACELLULAR MATRIX REVERSIBLY REGULATES MORPHOLOGY,PROLIFERATION AND COLLAGEN METABOLISM OF PERISINUSOIDAL STELLATE CELLS (VITAMIN A-STORING CELLS)

The three-dimensional structure of the extracellular substratum was found to regulate reversibly the morphology, proliferation and collagen synthesis of perisinusoidal stellate cells (lipocytes, i.e. fat-storing ‘Ito’ cells). On non-coated polystyrene and type I collagen-coated culture dishes, the cells spread well and extended their cellular processes. On the surface of type I collagen gels, the cells gathered and formed a mesh-like structure. However, in type I collagen gel where the cells were surrounded by type I collagen three-dimensionally, the cells extended their fine cellular processes and resembled the star-shaped stellate cells seenin vivo. The cell proliferation was more prominent in culture dishes coated with type I collagen or in polystyrene culture dishes than on or in type I collagen gels. The collagen synthesis was affected in the same manner. These data indicate that the nature and the three-dimensional structure of the extracellular matrix (ECM) can regulate morphology, proliferation and functions of the perisinusoidal stellate cells. In order to examine the reversibility of these regulations, we liberated cultured cells with trypsin or with purified bacterial collagenase and re-seeded them onto or into each substratum. The cells changed their shape, rate of proliferation and collagen synthesis according to each new substratum. These results indicate that the three-dimensional structure of ECM reversibly regulates the morphology, proliferation rate and functions of the perisinusoidal stellate cells.     

WHERE AM I? HOW A CELL RECOGNIZES ITS POSITIONAL INFORMATION DURING MORPHOGENESIS

Morphogenesis is an old, and one of the latest, fascinating fields in biological science and a huge number of papers on molecular mechanisms underlying it have been published. But most of the works and reviews on these mechanisms pertain to molecules of, as it were, the planning or design of morphogenesis, such as morphogens and homeodomain proteins. In this review, I will describe the function of extracellular matrix (ECM) and other cell adhesion molecules in morphogenesis as that of actual morpho-creating molecules, morphocreators, and discuss their roles as positional information-pertaining molecules.     

CELL POSITION-DEPENDENT RECIPROCAL FEEDBACK REGULATION OF TYPE I COLLAGEN GENE EXPRESSION IN CULTURED HUMAN SKIN FIBROBLASTS

In order to investigate possible cell positional effects on the gene expression of human dermal fibroblasts, the authors cultured the cells on non-coated polystyrene culture dishes, type I collagen-coated dishes, or collagen gels formed by type I collagen, or suspended them in type I collagen gels and measured collagen synthesis by the cells. The production rate of type I collagen was similar whether cells were cultured on non-coated polystyrene or on type I collagen-coated dishes, but it was suppressed significantly when the cells were placed within the collagen gel matrix. Time-dependent expression of genes for α1(I) and α2(I) collagen chains was measured by Northern blot analysis. A significant increase in mRNA levels for these chains was observed when the cells were cultured for three days on type I collagen-coated dishes or on collagen gels. On the other hand, a significant decrease in the mRNA levels was observed after 2 days and later, when the cells were cultured within type I collagen gel matrix. These results indicate that human dermal fibroblasts recognize their position on or in type I collagen (extracellular matrix) and respond by changing their expression patterns of type I collagen chain genes. The results of the kinetics of gene expression also suggest that upregulation and downregulation of type I collagen genes are controlled by different mechanisms.     

STIMULATION OF COLLAGEN SYNTHESIS IN RAT CARDIAC FIBROBLASTS BY EXPOSURE TO HYPOXIC CULTURE CONDITIONS AND SUPPRESSION OF THE EFFECT BY NATRIURETIC PEPTIDES

Synthesis of type I and type III collagens by rat cardiac fibroblasts was stimulated when the cells were cultured under 95% N₂/5% CO₂for one hour followed by incubation under normoxic conditions for 24 hours. The stimulative effect was attenutated by the presence of atrial natriuretic peptide (ANP, 10⁻⁶m) or brain natriuretic peptide (BNP, 10⁻⁶m) in the culture medium. Northern blot analysis indicated that α1(I) and α1(III) collagen mRNA levels were also increased by hypoxia, and decreased with the addition of ANP or BNP in a dose-dependent manner. These results indicate interaction between intracellular signals of a physical stimulus (hypoxic stress) and those of a chemical one (ANP or BNP) and demonstrate that both signals regulate collagen synthesis by cardiac fibroblasts at the levels of the mRNAs. The results also suggest that natriuretic peptides produced by cardiomyocytesin vivomay function as paracrine factors that play a role in the prevention of cardiac fibrosis in ischaemic heart diseases.