Density gradient characterization of the high density lipoproteins in cholesterol-fed hyper- and hyporesponding patas monkeys (Erythrocebus patas)

Diet-induced changes in high density lipoprotein (HDL) density and size were studied in patas monkeys. When the animals were switched from a moderate fat-low cholesterol diet to a high fat-high cholesterol (HFHC) diet, the plasma apoA-I levels increased initially in all of the animals. The apoA-I levels remained elevated in monkeys able to maintain their plasma cholesterol concentrations near basal levels (hyporesponders), but began to decrease in monkeys who became severely hypercholesterolemic (hyperresponders), reaching levels as low as 65-70% of their basal value by 24 weeks. The larger, lipid-rich HDL (HDL2) was shown by density gradient ultracentrifugation and gradient-PAGE (polyacrylamide gel electrophoresis) to be the HDL fraction responsible for these changes in apoA-I, completely accounting for the increase in apoA-I in hyporesponders and the decrease in apoA-I in hyperresponders. The HDL3 levels remained unchanged in hyporesponders but increased markedly in hyperresponders, partially compensating for the decrease of HDL2 in those animals. Gradient-PAGE showed the HDL3 to be heterogeneous, containing at least two populations of particles of the same density but differing significantly in size. The smaller of these HDL3 were most prominent in the HFHC-fed hyperresponders. These data show that nonhuman primate HDL is both physically and metabolically heterogeneous, and indicate that a high fat-high cholesterol diet-induced hypercholesterolemia severely depresses the HDL2 levels.     

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.     

Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance

The suitability of proteome-based strategies for the targeting of tumor-associated markers along with further analysis regarding their clinical significance were investigated in human renal cell carcinoma (RCC). The immunogenic protein expression profile of normal kidney and RCC cell lines was studied by proteome analysis combined with immunoblotting using sera from healthy donors and RCC patients, also termed PROTEOMEX. Employing this approach, a series of proteins reactive with either RCC patient sera and/or reactive with control sera were identified by microanalysis of tryptic peptides. Some of these candidate antigens represent members of the cytoskeletal family, such as cytokeratins, in particular cytokeratin 8, cytoskeletal tropomyosin, F-actin capping protein, gamma-actin, stathmin, tubulin-alpha, tubulin-beta and vimentin. The expression pattern and clinical significance of three of these antigens, namely cytokeratin 8, stathmin and vimentin, were further analyzed in a large series of surgically removed RCC lesions of distinct subtypes. A heterogeneous expression pattern of cytokeratin 8, stathmin and vimentin was demonstrated in the different RCC subtypes. All epithelial cells of the autologous normal kidney showed a strong cytokeratin 8 staining pattern, whereas they totally lack vimentin expression. Stathmin was expressed in 10% of tubule cells. In conclusion, PROTEOMEX could be employed for the identification of tumor-associated antigens of the cytoskeleton which are differentially expressed in RCC of distinct subtypes as well as in normal renal epithelium.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.