Tikva Vogel

Apolipoprotein E Protects Against Bacterial Lipopolysaccharide-induced Lethality A NEW THERAPEUTIC APPROACH TO TREAT GRAM-NEGATIVE SEPSIS

Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines. Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo. We now report that intravenously administered LPS strongly increases the serum levels of apoE. In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents. Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice. These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.

Method for recovering a purified animal growth hormone or polypeptide analog thereof from a bacterial cell

A method is provided for recovering a purified animal growth hormone or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, the corresponding naturally-occurring animal growth hormone from a bacterial cell in which the animal growth hormone or polypeptide analog has been produced by means of expression of a plasmid encoding the hormone or polypeptide analog which comprises: (a) disrupting the cell wall of the bacterial cell in a buffered neutral pH solution so as to produce a lysate containing precipitated hormone or polypeptide analog; (b) recovering the resulting precipitated hormone or polypeptide analog; (c) suspending the precipitated hormone or polypeptide analog so recovered in distilled water; (d) treating the resulting precipitate-containing suspension with a sodium hydroxide solution having an alkaline pH of about 11.8 so as to solubilize the precipitate and thus the hormone or polypeptide analog contained therein; (e) separating the solubilized hormone or polypeptide analog from other soluble components by gel filtration chromatography; and (f) subjecting the hormone or polypeptide analog thus separated to ion exchange chromatography to purify the hormone or analog and thereby recover purified hormone or polypeptide analog.

A synthetic heparin-mimicking polyanionic compound binds to the LDL receptor-related protein and inhibits vascular smooth muscle cell proliferation.

A synthetic heparin‐mimicking polyaromatic anionic compound RG‐13577 (polymer of 4‐hydroxyphenoxy acetic acid and formaldehyde ammonium salt, Mr∼5800) exhibits specific binding to vascular smooth muscle cells (SMCs) and inhibits their proliferative response to growth promoting factors. Receptor binding of 14C‐RG‐13577 was efficiently competed by apolipoprotein E3 (apoE), lactoferrin, and the LRP (LDL receptor‐related protein) receptor associated 39 kDa protein (RAP). Unlike cell surface binding of apoE, binding of RG‐13577 to SMCs was not affected by heparin, heparan sulfate degrading enzymes, or low density lipoprotein (LDL). Moreover, wild‐type and heparan sulfate‐deficient Chinese hamster ovary (CHO) cells, as well as normal‐ and LDL receptor negative‐ human skin fibroblasts bind RG‐13577, but not apoE, to a similar extent. On the other hand, homozygous mouse embryonic fibroblasts deficient in the LDL receptor‐related protein (LRP) expressed a markedly reduced binding of RG‐13577 as compared to normal mouse embryonic fibroblasts. These results indicate that RG‐13577 and related compounds bind to the LRP receptor on the surface of vascular SMCs. Addition of lactoferrin to cultured SMCs protected the cells against the antiproliferative effect of compound RG‐13577, suggesting that this inhibition is mediated by RG‐13577 binding to LRP receptors on the SMC surface. Altogether, we have identified a series of synthetic polyaromatic anionic molecules that exhibit specific binding to LRP and therby exert an antiproliferative effect on vascular SMCs. These compounds are applied to suppress SMC proliferation associated with restenosis and accelerated atherosclerosis. J. Cell. Biochem. 81:114–127, 2001.

Effect of N-terminal modified analogs of growth hormone on collagen synthesis in avian skin fibroblasts

Human growth hormone (hGH) inhibits alpha 1(I) collagen gene expression in cultured avian skin fibroblasts resulting in a decrease in the amount of collagenase-digestible proteins (CDP) in the medium. In addition, a synergism exists between GH and insulin-like growth factor-I (IGF-I) in their effect on CDP. Four N-terminal modified hGH analogs were tested for their ability to affect collagen metabolism in these cells. The truncated analog Des-7 hGH(R8M, D11A) was found to be a strong antagonist of the hGH-induced inhibition of the collagen synthesis but by itself did not inhibit collagen alpha 1(I) gene expression or modify the CDP appearance in the medium. Some synergism between Des-7 hGH and IGF-I was observed. The analog Met-hGH(R19H, L20P), in which Arg19 was replaced by histidine, and Leu20 by proline was only partially potent compared with the native hormone in causing inhibition of collagen gene expression, in attenuating CDP appearance in the medium, and in antagonizing hGH. However, this analog was as potent as hGH in its ability to synergize with IGF-I. The importance of His18 was assessed by testing the response to Met-hGH(H18D), in which His18 was replaced by Asp, and to Met-hGH(H18Q), in which His18 was replaced by glutamine (as in chicken GH sequence). Substitution of His18 by a negatively charged amino acid abolished all the hormone activities tested whereas substitution with glutamine restored only part of the activity.(ABSTRACT TRUNCATED AT 250 WORDS)