Nucleotide sequences of RNA codons have been investigated recently by directing the binding of C'4-AA-sRNA to ribosomes with trinucleotides of defined base sequence. The template activities of 19 trinucleotidest have been described and nucleotide sequences have been suggested for RNA codons corresponding to 10 amino acids.'-5 In this report, the template activities of 26 additional trinucleotides are described and are related to the general nature of the RNA code. Materials and Methods.-Components of reactions: E. coli W3100 ribosomes and sRNA were prepared by modifications of methods described previously.6' Each C14-aminoacyl-sRNA was prepared in the presence of 19 C"2-amino acids. The assay for ribosomal bound C'4-AA-sRNA and components of reaction mixtures have been described.' The characteristics and amounts of labeled AA-sRNA not described' are shown in Table 1. Synthesis and characterization of oligonucleotides: ApG and UpG were obtained from a T-1 ribonuclease digest of RNA, and ApA was prepared by chemical synthesist 10 ApC, ApU, CpA, CpG, GpA, and GpC were obtained from Gallard Schlessinger Corp., but required extensive purification prior to use. GpGpU was obtained by digesting poly UG with pancreatic RNase A; treatment with alkaline phosphatase to remove terminal phosphate groups from the degradation products; and isolation by procedures similar to those described for GpUpU.2 The remaining trinucleotides were synthesized frdm the appropriate dinucleoside monophosphate, using either TABLE 1 RADIOACTIVE AMINoAcYLsRNA PREPARATIONS* C14 or HL-AA-sRNA Added to Each Reaction Specific #sjmoles of C14-Origin of Radioactive radioactivity or H'-Amino sRNA E. coli amino acidt jsc/pmole A250 units acid accepted straint LYS 240 0.25

Reduced growth-factor requirement of keloid-derived fibroblasts may account for tumor growth (

Extracellular matrix hyaluronan is prominent during wound healing, appearing at elevated levels early in the repair process. It is prevalent throughout the course of fetal wound healing, which is scar-free, but decreases late in adult wound repair, that is often marked by scarring. To determine whether aberrant hyaluronan metabolism is associated with the excessive scarring that characterizes keloids, cultured fibroblasts derived from keloids and from the dermis of normal human skin and scar were compared. Levels of hyaluronan in 48 h conditioned media of keloid-derived cultures were significantly lower than in cultures of normal skin and scar fibroblasts. Profiles of hyaluronan polymer size were comparable in these two cell types, suggesting that excessive hyaluronan degradation was not involved. Hydrocortisone decreased hyaluronan levels approximately 70% in the conditioned media of both keloid and normal fibroblasts. Diminished hyaluronan accumulation in keloid-derived cells compared with normal fibroblasts was also observed in an in vitro wound healing model. Histolocalization of hyaluronan in keloids, normal skin, and scar samples confirmed the biochemical observations that the dermis of keloids, which comprises most of the scar tissue, contained markedly diminished levels of hyaluronan. Alterations in hyaluronan in the epidermis overlying keloids, however, were also observed. A modest increase in hyaluronan staining intensity was observed in the epidermis of keloids, as well as changes in the patterns of distribution within the epidermis, compared with that in normal skin and scar. Increased hyaluronan was present in the granular and spinous layers of the keloid epidermis Abnormalities are present apparently in both the overlying epidermis as well as in the dermis of keloids. Aberrations in signaling between keloid stroma and keloid epidermis may underlie abnormalities that contribute to the excessive fibrosis characteristic of these lesions.

Keloids are benign collagenous tumors that occur during dermal wound healing in genetically predisposed individuals. The lesions are characterized by over-proliferation of fibroblasts, some leukocyte infiltration, and prolonged high rates of collagen synthesis. To determine whether leukocyte chemoattractants or chemokines are participating in this disease process, immunohistochemical staining for the CXC chemokine, MGSA/GROα, and its receptor, CXCR2, was performed on tissue from keloids, hypertrophic scars and normal skin. Immunoreactive MGSA/GROα was not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts and lymphocytes in nodular areas of the keloid samples. This staining positively correlated with the degree of inflammatory infiltrate in the lesions. Keloids, but not hypertrophic scars or normal dermis, also exhibited intensive immunoreactivity for the CXCR2 receptor in endothelial cells and inflammatory infiltrates with occasional staining of myofibroblasts. In contrast, cultured fibroblasts from either keloids or normal skin did not express detectable amounts of mRNA for MGSA/GRO or CXCR2, although interleukin-1 strongly induced MGSA/ GRO mRNA in both cell types. Interleukin-1 induction of MGSA/GRO was inhibited by glucocorticoid in normal and keloid fibroblasts, and the effect was more pronounced in keloid fibroblasts. This event was not correlated with inhibition of nuclear activation of NF-κB, AP-1 or Sp1, and might therefore be mediated by another mechanism such as decreased mRNA stability or transcriptional repression through the glucocorticoid response element in the MGSA/GRO promoter. Data from in vitro wounding experiments with cultured normal and keloid fibroblasts indicate that there were no significant differences in MGSA/GRO or CXCR2 receptor levels between normal and keloid fibroblasts. We also show that cultured keloid fibroblasts exhibit a delayed wound healing response. We postulate that the inflammatory component is important in development of keloid lesions and chemotactic cytokines may participate in this process.Keloids are benign collagenous tumors that form in the reticular layer of the dermis during a prolonged wound healing process in persons with a genetic predisposition. 1 to be restricted to dermal wound healing, because other growth or connective tissue abnormalities are not frequently reported in keloid patients. The disorder may be genetically heterogeneous, with both dominant and recessive modes of inheritance having been reported. Genetically susceptible individuals form keloids after wounding. Abnormalities in cell migration, proliferation, inflammation, synthesis and secretion of extracellular matrix proteins and cytokines, and remodeling of the wound matrix have all been described in keloids. 3,4 Black patients with keloids often exhibit increased activity of fibrogenic cytokines 5,6 as well as an altered cytokine profile. 7 The exaggerated wound healing process in keloids appears to be due in part to loss of glucocorticoid supp...

Keloids arise as benign connective tissue masses at sites of injury in genetically predisposed individuals. In addition to excessive collagen accumulation, there is biochemical and histologic evidence of elastic tissue. Previous studies showed that glucocorticoid regulation of collagen synthesis differs in fibroblasts from normal adult dermis and keloids. To define further the abnormal regulation of matrix synthesis in keloid fibroblasts, we examined glucocorticoid regulation of elastin synthesis. The basal level of elastin synthesis was significantly higher in keloid than in normal cells, and hydrocortisone reduced synthesis of elastin and elastin mRNA in normal but not in keloid fibroblasts. We had shown previously that fibroblasts from fetal dermis resembled keloid fibroblasts in glucocorticoid regulation of growth and collagen synthesis. In this study, glucocorticoids failed to down-regulate elastin synthesis in fetal cells that had not differentiated to produce normal levels of elastin, whereas fetal cells with normal elastin production exhibited glucocorticoid down-regulation. Abnormal regulation in keloid cells was independent of cell density and was confined to fibroblasts cultured from the keloid nodule. These findings reinforce the conclusion that a matrix-regulatory pathway is deranged in these focal lesions. Coordinate down-regulation of collagen and elastin by hydrocortisone in normal adult dermal fibroblasts and the failure of hydrocortisone to down-regulate synthesis of either protein in keloid cells support the existence of common elements in the regulatory pathways of these two matrix proteins.