The role of the degree of oligomerization in the structure and function of human surfactant protein A (SP-A) was investigated using a human SP-A1 mutant (SP-A1 ⌬AVC,C6S ), expressed in mammalian cells, resulting from site-directed substitution of serine for Cys 6 and substitution of a functional signal peptide for the cysteine-containing SP-A signal sequence. This Cys 6 mutant lacked the NH 2 -terminal Ala ؊3 -Val ؊2 -Cys ؊1 (⌬AVC) extension present in some SP-A1 isoforms. SP-A1⌬AVC,C6S was assembled exclusively as trimers as detected by electron microscopy and size exclusion chromatography. Trimeric SP-A1 ⌬AVC,C6S was compared with supratrimeric SP-A1, which is structurally and functionally comparable to the octadecameric protein isolated from human lung lavages. SP-A1 ⌬AVC,C6Sshowed reduced thermal stability of the collagen domain, studied by circular dichroism, and increased susceptibility to trypsin degradation. The T m was 32.7°C for SP-A1 ⌬AVC,C6S and 44.5°C for SP-A1. Although SP-A1 ⌬AVC,C6S was capable of binding to calcium, rough lipopolysaccharide, and phospholipid vesicles, this mutant was unable to induce rough lipopolysaccharide and phospholipid vesicle aggregation, to enhance the interfacial adsorption of SP-B/SP-C-surfactant membranes, and to undergo self-association in the presence of Ca 2؉ . On the other hand, the lack of supratrimeric assembly hardly affected the ability of SP-A1 ⌬AVC,C6S to inhibit the production of tumor necrosis factor-␣ by macrophagelike U937 cells stimulated with either smooth or rough lipopolysaccharide. We conclude that supratrimeric assembly of human SP-A is essential for collagen triple helix stability at physiological temperatures, protection against proteases, protein self-association, and SP-A-induced ligand aggregation. The supratrimeric assembly is not essential for the binding of SP-A to ligands and anti-inflammatory effects of SP-A.

A protein was isolated from plasma of partially (70%) hepatectomized rats that, injected in mice, increases the uptake of [3H]thymidine by liver DNA by 200-300% over that by injected control saline. The purification procedure consists essentially of three chromatography steps, employing Sephadex G-75, DEAE-cellulose and hydroxyapatite. The hepatic promoter (HP) preparation shows a single band in SDS/polyacrylamide (15%)-gel electrophoresis (silver stained), with an Mr of 64 000; its activity is suppressed by trypsin or pepsin and is unaffected by deoxyribonuclease or ribonuclease. On injection into mice (150 ng/mouse), it increases the mitotic index of the liver. It shows organ-specificity, acting on liver but not on spleen, kidney, lung or brain. In primary liver cultures, it produces an increase in uptake of [3H]thymidine into DNA in the range 1-10 ng/ml. In this system in vitro, it increases the uptake of 22Na+ immediately after addition.

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Tacrolimus (FK506) is a hydrophobic immunosuppressive agent that rapidly penetrates the plasmatic membrane and inhibits the signal transduction cascade of T lymphocytes. The objective of this study was the characterization of liposomal FK506 with surfactant-like phospholipids to be administered intratracheally after lung transplantation or in inflammatory lung diseases. We evaluated the optimal incorporation of FK506 in dipalmitoylphosphatidylcholine (DPPC) and DPPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) monolayers and bilayers and the effects of FK506 on the physical properties of DPPC and DPPC/POPG (8:2 w/w) vesicles. In addition, we assessed the immunosuppressive effects of surfactant-like phospholipid vesicles containing different amounts of FK506 on T-cell proliferation and interleukin 2 production. From surface pressure measurements of FK506/DPPC and FK506/DPPC/POPG mixed monolayers, we determined that FK506 was embedded into these monolayers up to an FK506 concentration of about 0.4 mol %. Beyond this concentration, FK506 was not quantitatively incorporated into the monolayer, suggesting possible concentration-dependent aggregation of tacrolimus. The incorporation of FK506 into DPPC monolayers, at concentrations

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We have reported the purification and characterization of a protein that behaves as a liver growth factor, showing activity either in vivo or in vitro [Díaz-Gil et al. (1986) Biochem. J. 235, 49-55]. In the present paper, we identify this liver growth factor (LGF) as an albumin-bilirubin complex. This conclusion is supported by the results of chemical and spectroscopic characterization of this protein as well as by experiments in vivo. Incubation of albumin isolated from normal rats with bilirubin/albumin molar ratios (r) resulted (when r = 1 or 2) in a complex with liver DNA synthesis promoter activity identical with that of LGF. The exact amount of bilirubin bound to albumin was assessed by fluorescence and c.d. spectra. This albumin-bilirubin complex showed the same dose-dependence profile as LGF either at low or high dose of protein injected per mouse. Both LGF and albumin-bilirubin complex produced similar increases in the mitotic index of mouse hepatocytes in vivo. A new mechanism for the onset of the hepatic regenerative process is proposed.

Renal growth factor activity was extracted from plasma of adult uninephrectomised rats and partially purified by gel filtration and anion-exchanger FPLC. It induced a maximal stimulation of mouse DNA synthesis in vivo at 1.75 micrograms/mouse. In addition, renal growth factor was found to maximally stimulate DNA synthesis in LLC-PK1 cells at 150 ng/ml. This maximal response was then found to decrease with higher doses of renal growth factor, in vivo and in vitro. The apparent molecular weight of renal growth factor was estimated to be 17K-22 K by gel filtration. It was found to be resistant to heat and to trypsin, but labile to reduction with dithiothreitol.

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