The cytosolic pathway of L-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase.

Saccharomyces cerevisiae accumulates L-malic acid but not only minute amounts of fumaric acid. A 13C-nuclear magnetic resonance study following the label from glucose to L-malic acid indicates that the L-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to L-malic acid is responsible for the L-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to L-malic but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards L-malic acid; the Km for L-malic acid is very high indicating that L-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more L-malic acid). (c) According to 13C NMR studies there is no interconversion of cytosolic L-malic and fumaric acids.

Thrombospondin and in vivo angiogenesis induced by basic fibroblast growth factor or lipopolysaccharide.

PURPOSE: To reexamine the possible effect of human thrombospondin on in vivo angiogenesis. METHODS: In vivo angiogenesis in the rabbit cornea was induced by implants of Elvax-40 sequestering human recombinant basic fibroblast growth factor (bFGF) or bacterial endotoxin lipopolysaccharide (LPS). Implants sequestering various concentrations of thrombospondin were examined for their ability to induce angiogenesis and also for their possible influence on the angiogenic potential of bFGF- or LPS-sequestering implants. RESULTS: Constant and reproducible angiogenic stimuli were obtained with implants sequestering 250 ng or more of bFGF or 100 ng or more of LPS. Implants sequestering 500 ng of thrombospondin induce very little clinical change but larger concentrations induce infiltration of leukocytes and a mild angiogenic stimulus. Combination of thrombospondin implants with bFGF or LPS implants enhanced the angiogenic response to either of these factors. The thrombospondin enhancing effect was more prominent when LPS was the stimulating factor. Histologic examination of the tested corneas disclosed that the LPS angiogenic stimulus follows the influx of leukocytes. Conversely, the bFGF angiogenic stimulus appears to be associated with the proliferation of stromal keratocytes and corneal epithelial cells. The thrombospondin angiogenic enhancing effect on both the LPS and bFGF stimuli was correlated with an increased infiltration of polymorphonuclear cells. CONCLUSION: In this system, thrombospondin enhanced the in vivo angiogenic process induced by bFGF or LPS. This enhancement appears to be associated with an in vivo activation and chemotactic effect on the polymorphonuclear cells.

Effect of Cyclosporin A, Rapamycin, and FK-506 on corneal epithelial cells and lymphocyte proliferation.

The proliferation of corneal epithelial cells in vitro is relatively resistant to the addition of immunomodulating drugs to the cultures. Cyclosporin A, FK-506, and Rapamycin cause minimal or no inhibition of corneal epithelial cells proliferation in concentrations of up to 500 ng/ml. At concentrations of 5000 ng/ml, all drugs induce an inhibitory effect. In this system, Rapamycin induces the strongest inhibition in most cases. Nonetheless, a residual activity of more than 50% is recorded even at the highest concentrations. On the lymphocytes, both FK-506 and Rapamycin show marked inhibitory effects at 0.5 ng/ml, while CsA shows significant inhibition only at the level of 50 ng/ml or higher. These data can be interpreted to indicate a possible large therapeutic window (marked inhibition of lymphocyte proliferation at drug concentrations which do not have any effect on epithelial cell proliferation) of these drugs for topical use in external ocular diseases.

The role of cytokines in angiogenesis.

Using the corneal model of neovascularization developed in their laboratory, the authors investigated the angiogenic potential of interleukin-1 (IL-1α and ß), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor (TNFα), transforming growth factor ß (TGFß), granulocyte macrophage-colony stimulating factor (GM-CSF), and interferon (IFN(γ) and IFN(γ)). Various concentrations of the tested cytokine were sequestered into Elvax-40 and implanted at 2.5 mm from the limbus within the corneal stroma of the rabbit eye. Three distinct groups of cytokines could be observed according to their angiogenic potential in this system. IL-1α was by far the most potent stimulator of neovascularization, inducing this process at concentrations as low as one nanogram per implant. TGFß, TNFα and GM-CSF induced significant neovascularization only at concentrations of 500 nanograms per implant. IL-2, IL-6, IL-8, IFNα and IFN(γ) did not induce any significant neovascularization at the concentration tested (0.5 µg to 10 µg per implant).

Influence of interferons corneal angiogenesis induced by basic fibroblast growth factor and lipopolysaccharide.

A controlled and reproducible angiogenic stimulus was induced in the rabbit cornea by Elvax-40 implants sequestering either 500 ng of lipopolysaccharide (LPS) or 500 ng of basic fibroblast growth factor (bFGF). The effect of IFNα and IFN(ß) on angiogenesis was studied by inserting implants sequestering 500 ng of these cytokines (approximately 10(4) Units/implant) adjacent to the LPS or bFGF implants. Interferon-α or γ did not inhibit the bFGF-induced angiogenesis, and in most of these experiments an enhancing effect was observed. This enhancement was not statistically significant. The LPS-induced angiogenesis, however, was slightly inhibited in the presence of either interferon-α or γ but these differences were not statistically significant with p=0.1 only. Both cytokines were non angiogenic and there was no detectable difference between them regarding their effect on the angiogenic process induced by either bFGF or LPS.