Loren Pickart

Stimulation of collagen synthesis in fibroblast cultures by the tripeptide‐copper complex glycyl‐L‐histidyl‐L‐lysine‐Cu2+

Glycyl‐L‐histidyl‐L‐lysine (GHK) is a tripeptide with affinity for copper(II) ions and was isolated from human plasma. This peptide appears to play a physiological role in wound healing. We report the stimulating effect of GHK‐Cu on collagen synthesis by fibroblasts. The stimulation began between 10−12 and 10−11 M, maximized at 10−9 M, and was independent of any change in cell number. The presence of a GHK triplet in the α2(I) chain of type I collagen suggests that the tripeptide might be liberated by proteases at the site of a wound and exert in situ healing effects.

Fatty acids, fibrinogen and blood flow: A general mechanism for hyperfibrinogenemia and its pathologic consequences

Plasma fibrinogen is elevated in various stressful states and conditions in which active mobilization of free fatty acids (FFA) occurs. Reduction of plasma FFA by an assortment of hypolipidemic drugs is consistently followed by a decrease in the accompanying hyperfibrinogenemia. A direct link between FFA and fibrinogen has been demonstrated in animals, and in experiments employing incubated liver slices. Based on these clinical and experimental observations, we postulate that hepatic fibrinogen synthesis is stimulated by FFA. Since fibrinogen is a major determinant of whole blood viscosity, erythrocyte aggregation, and sludging of red cells in terminal and pre-terminal blood vessels, we propose that microcirculatory blood flow may be impaired in the presence of chronically elevated plasma FFA levls. Consequently, hypolipidemic drugs may be effective in prevention of circulatory complications associated with FFA-induced hyperfibrinogenemia.

Purification of Growth-Promoting Peptides and Proteins, and of Histones, by High Pressure Silica gel Chromatography

A rapid method for the purification of histones and a variety of growth-promoting proteins and peptides by chromatography on silica gel has been developed. The isolation of the growth-promoting components of serum has been hampered by excessive losses associated with the use of water-based purification mens in acidic methanol-H2O solutions (eg. insulin, albumin, the somatomedins) provides a basis for purification on high-pressure silica gel columns, while peptides and histones can be purified in similar solvents. After column chromatography, the solvent is removed by flash-evaporation, or the protein may be precipitated directly from the solvent by neutralization of the pH and the addition of ethanol. The retention of biological activity (eg. somatomedin-C binding to insulin receptors and cell-growth stimulation) and recovery are excellent.

Growth-modulating human plasma tripeptide: relationship between molecular structure and DNA synthesis in hepatoma cells.

H-glycylhistidyllysine-OH (GHL) is a tripeptide found at approximately 200 ng/ml in human plasma in association with the albumin and o-globulin fractions [ 1,2 J. When added to culture medium at 1 S-100 ng/ml, synthetic GHL promotes the proliferation of hepatoma cells ]2,3], lymphocytes [4] and T-strain mycoplasma [5]; maintains the viability of normal hepatocytes [2,3], eosinophils [6], and macrophages [7]; inhibits the growth of glial cells [8]; and supports the growth and differentiation of neurons ]8] and ascaris larvae [9]. At higher concentration, the tripeptide inhibits L929 cell growth (500 ng/ml) [lo] and maintains the viability of mast cells (20 pg/ml) during degranulation tests [ 1 I]. In general, GHL acts to reduce or eliminate the amount of serum required for culture of the various cell types or organisms [2-4,6-9,111. Although the mechanism of action of GHL is unknown, the tripeptide is co-isolated with copper and iron [ 12,131, and acts synergistically with these transition metals to stimulate the growth and metabolism of hepatoma cells maintained in grows-l~it~g amounts of serum [ 12,131. In this paper we describe the effects of 9 synthetic analogs of GHL on DNA synthesis in hepatoma cell culture. The results indicate that 2 structural features are involved in the bioactivities of GHL: (1) the histidyllysyl linkage and (2) a glycyl residue in either terminal position.

Hyperfibrinogenemia and polycythemia with intrauterine growth retardation in fetal lambs

Plasma concentrations of albumin and fibrinogen and arterial hematocrits were determined during the last third of gestation in growth-retarded and control fetal lambs. The mean fetal plasma albumin concentration increased slightly as term approached and was not significantly different in the two groups. The mean plasma fibrinogen concentration did not change in the control fetuses, but was significantly elevated in the growth-retarded fetuses, as was the mean arterial hematocrit. The theoretical implications of these findings relative to capillary blood flow are discussed.

Alteration in biological properties of human albumin during storage

Abstract The effects of human albumin preparations on oxidative energy metabolism and lipid svnthesis were investigated in rat liver slices incubated with sodium [1-14C]acetate as precursor. Labeled CO2 production and incorporation of precursor into the major lipid classes was increased 2 to 3-fold by fresh preparations of albumin (fraction V), and by defatted fraction V, whereas highly purified cystalline albumin was less active. Albumin preparations from various commercial suppliers varied widely in activity. Activity of fraction V was preserved during storage at −20°C, and gradually lost at +3°C in the course of 1 year. In contrast, defatted fractions rapidly lost activity in storage at both temperatures. After 1 year in storage at +3°C, albumin preparations became inhibitory to CO2 production and lipid synthesis. The results suggest that commercial albumin used in metabolic studies, and in clinical situations may have unpredictable or undesirable effects related to state of purity and storage conditions of the protein.

Fibrinogen and albumin synthesis during fetal development in mice.

Abstract The hepatic synthesis of fibrinogen and albumin in mice during fetal development (12 to 20 days gestation) was determined. Fetal hepatic synthesis of fibrinogen matures relatively rapidly and reaches adult levels by the 16th day of gestation. In contrast, albumin production develops more slowly, attaining 80% of adult values immediately prior to birth at 20 days. Autoradiography of fetal hepatocytes cultured in agar established that, in the earliest obtainable hepatocytes (12 days gestation), all cells were producing fibrinogen but a fraction of hepatocytes (about 8%) were not yet producing albumin.

Improved recovery of somatomedin activity from plasma by prolonged formic acid extraction

Recovery of somatomedin activity from plasma fractions was increased approximately 5-fold by prolonged extraction of isolates with formic acid. Extraction for 72 hours enhanced the yields of active peptides in the 6,000, 8,000 and > 20,000 dalton range. Yields of smaller peptides were similar following short (2 hrs) and long (72 hrs) extraction periods. Active fractions obtained after prolonged extraction possessed sulfation activity and insulin-receptor affinity comparable to somatomedin isolated during short extraction with formic acid.

EFFECTS OF TREATMENT WITH EMULSIFIED FAT SUSPENSION (IL) ON PIASMA FIBRINOGEN (F)

A rise in circulating triglyceride or free fatty acids (FFA) is often associated with increased F, and recent data indicate that FFA may be involved in regulation of F synthesis. F was measured in young rats allowed access to regular chow while treated with a 10% suspension of IL (3 to 6 g/kg/day) administered P.O., by continuous i.v. infusion, or by serial i.p. injection. In parallel experiments, animals also received injections of heparin, 500 U (H). Controls were given saline by the corresponding route plus chow, or chow only. Animals were bled by aortic puncture at 48 hrs. F averaged 266 ± 0.14 (SEM) mg% in untreated and p.o. controls, 549 ± 0.35 mg% in i.v. controls, and 349 ± 0.54 rag% in i.p. controls. These differences in control values may reflect stress effects of confinement or injection (FFA release?). F in rats receiving IL or IL + H, respectively, increased by 21% (N.S.) and 71% (p < 0.01) in the p.o. series; 70% (p< 0.001) and 63% (p < 0.001) in the i.v. series; and 87% (p < 0.001) in the i.p. series. Thus, F increased significantly when fat was infused directly (i.v.) or indirectly (i.p.) into the circulation, or when H was administered with IL p.o. In contrast, IL administered p.o. without H had no effect on F. These results indicate that i.v. therapy with IL may be followed by a significant rise in F, with potential increase in blood viscosity and decrease in peripheral blood flow in polycytnanic or severely stressed infants.