Opposing functions of Ki- and Ha-Ras genes in the regulation of redox signals.

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response [1-2]. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex [3]. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable [4]. We show that Ki- and Ha-Ras genes differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription.

Presence of CuZn superoxide dismutase in human serum lipoproteins.

It has previously been demonstrated that CuZn-superoxide dismutase (SOD) is secreted by several human cell lines. This suggests that the circulating enzyme derives from both hemolysis and peripheral tissues as a result of cellular secretion. In the present report, we evaluated the presence of CuZn-SOD in human serum lipoproteins by both enzyme-linked immunosorbent assay and Western blot analysis of immunoprecipitated lipoprotein samples. The distribution of CuZn-SOD activity among the different lipoprotein fractions was also determined by the xanthine/xanthine oxidase method. The results demonstrated that CuZn-SOD is noticeably present in serum lipoproteins and mainly in low and high density lipoproteins (LDL and HDL). Moreover, experiments performed by incubating CuZn-SOD with a lipid emulsion and subsequent separation of the lipid fraction by ultracentrifugation showed that this enzyme associates in a saturable manner with lipids. The CuZn-SOD bound to LDL and HDL could exert a physiological protective role against oxidative damage of these lipoprotein classes that carry out a crucial role in the cholesterol transport.

Free fatty acids modulate LDL receptor activity in BHK-21 cells.

It has been shown that dietary fatty acids affect serum low density lipoprotein (LDL) levels, but the mechanism responsible for this effect is still under debate. Here we investigate the effect of different free fatty acids on LDL receptor activity in BHK-21 cells. These cells possess a classical LDL receptor strongly regulated by substances like 25-OH-cholesterol or lovastatin. Preincubation of cells for 24 h with both oleic (cis 18:1) and its trans counterpart, elaidic acid, enhanced 125I-LDL binding, internalization and degradation, being oleic acid more effective than elaidic acid. Among polyunsaturated fatty acids (PUFA) of the n-6 series arachidonic acid (20:4) enhanced LDL receptor activity more than linoleic acid (18:2), and among PUFA of the n-3 series docosahexaenoic (22:6) and eicosapentaenoic acids (20:5) were more effective compared to alpha-linolenic acid (18:3). Conversely, preincubation of cells with saturated fatty acids, palmitic (16:0) and stearic (18:0) acids, decreased binding, internalization and degradation of 125I-LDL. Scatchard analysis of binding data obtained with palmitic and oleic acids showed that these two fatty acids affect LDL receptor number without altering receptor affinity. The regulatory effect of free fatty acids on LDL receptor activity in BHK-21 cells is consistent with the hypothesis that the ability of fatty acids to modulate LDL-cholesterol levels in vivo is mediated, at least in part, by an action on receptor-dependent uptake of LDL.

Secretion and increase of intracellular CuZn superoxide dismutase content in human neuroblastoma SK-N-BE cells subjected to oxidative stress.

CuZn superoxide dismutase (SOD) secretion was detected in media of [35S]cysteine-labeled human neuroblastoma SK-N-BE cells precipitated with antihuman CuZn SOD antibodies. The ability of Fe2+/ascorbate oxidative stress to induce CuZn SOD in SK-N-BE cells was evaluated by Western blot analysis. The results showed that, like human hepatocarcinoma cells and human fibroblasts, SK-N-BE cells secrete CuZn SOD. In addition, the CuZn SOD concentration was higher in cells subjected to oxidative stress than in unstressed cells. The secretion of CuZn SOD and the ability of Fe2+/ascorbate to increase its protein content in SK-N-BE cells indicates that this enzyme protects the brain from damage induced by oxidative stress.

Inhibitors of Ras farnesylation revert the increased resistance to oxidative stress in K-Ras transformed NIH 3T3 cells.

Tumor resistance to oxidative stress prevents the efficacy of cancer therapy based upon a free radical-mediated mechanism. K-ras transformed NIH 3T3 cells (E32-4-2) showed, under oxidative stress, reactive oxygen species (ROS) levels 10-fold lower and lipid peroxide levels 56% lower, compared to their nontransformed counterpart. Since p21(ras) activity depends upon farnesylation, we tested the effect of the inhibitors of farnesylation lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid on susceptibility to oxidative stress in these cells. Preincubation of cells for 24 h with 10 microM lovastatin resulted in a 10-fold increase of ROS levels and a 50% increase of lipid peroxide levels measured under pro-oxidant conditions. Similarly, preincubation of cells with 100 microM (alpha-hydroxyfarnesyl) phosphonic acid for 24 h enhanced stress-induced levels of either ROS (7.5-fold) or lipid peroxides (33%). The effect of lovastatin and (alpha-hydroxyfarnesyl) phosphonic acid is specifically due to their ability to inhibit p21(ras) activity. In fact, inhibition of p21(ras) by transfecting E32-4-2 cells with the transdominant negative mutant of H-ras (L61, S186) led, analogously to lovastatin or (alpha-hydroxyfarnesyl) phosphonic acid treatment, to a strong increase of stress-induced ROS levels. These results suggest that farnesylation inhibitors could be used as an adjuvant therapy to improve the tumoricidal effect of cancer treatment based upon free-radical production in ras-dependent tumors.

Evidence for secretion of cytosolic CuZn superoxide dismutase by Hep G2 cells and human fibroblasts.

The role so far ascribed to intracellular CuZn superoxide dismutase is that of an intracellular scavenger of oxygen radicals. However, other functions of cytosolic CuZn superoxide dismutase have been hypothesized. For example, CuZn superoxide dismutase incubated with rat hepatocyte cells in culture inhibits 3-hydroxy-3methylglutaryl CoA reductase, thereby reducing cholesterol synthesis. We recently demonstrated the presence of surface membrane receptors for CuZn superoxide dismutase, suggesting possible autocrine or paracrine activities. The aim of the present study was to investigate whether cytosolic CuZn superoxide dismutase can be secreted by human hepatocarcinoma and fibroblast cells lines. Proteins in human hepatocellular carcinoma (Hep G2) cells and human fibroblasts were biosynthetically labelled with [35S]-cysteine; then cell lysates and media were immunoprecipitated with rabbit polyclonal anti-human CuZn superoxide dismutase antibodies and separated by 12% polyacrylamide gel electrophoresis. Both Hep G2 cells and human fibroblasts produce and secrete CuZn superoxide dismutase which was detectable in cells and medium as a single protein band with the same electrophoretic mobility as human erythrocyte CuZn superoxide dismutase. These data suggest that CuZn superoxide dismutase, an enzyme thus far considered to be located exclusively intracellularly is secreted by at least two cell lines. This is consistent with autocrine or paracrine roles for CuZn superoxide dismutase.

Heterogeneity of DNA and RNA in Hunter patients.

Genomic DNA and total RNA from lymphoblasts of nine unrelated Italian patients affected with Hunter syndrome were analyzed using a human cDNA clone coding for the lysosomal enzyme iduronate-2-sulphatase (IDS). Southern blot analysis resulted in patterns similar to the normal control for seven of the patients analyzed; an aberrant pattern was observed in two patients (F.N. and P.D.), suggesting deletions/rearrangement in the IDS gene. Northern blot analysis showed in seven patients, a pattern similar to the normal control; for patients F.N. and P.D. the pattern was atypical, i.e., normal RNA species were absent whereas two different transcripts occurred. These data confirm the heterogeneity of the molecular defects causing Hunter disease.

Biochemical diagnosis of mucopolysaccharidoses: experience of 297 diagnoses in a 15-year period (1977-1991).

We report the results over 15 years (1977-1991) for biochemical diagnoses of patients referred from throughout Italy and suspected of having a mucopolysaccharidosis. Of these, 147 patients were diagnosed as being homozygous or hemizygous for a specific lysosomal enzyme deficiency; 74 pregnancies at risk were monitored in their families; 76 heterozygote diagnoses were performed on their relatives, with a total of 48 positive diagnoses. We also report the analysis of genomic DNA from 11 unrelated Italian Hunter patients, using pc2S15 probe. DNA from two patients, digested with Pst-I, showed a variant pattern of hybridization caused by deletion or rearrangement of the gene.

Cell-to-cell contact between normal fibroblasts and lymphoblasts deficient in lysosomal enzymes.

Human lymphoblasts deficient in iduronate sulfatase or in alpha-N-acetylglucosaminidase acquire discrete levels of enzyme activity after co-culture with human normal skin fibroblasts. This occurs by direct cell-to-cell contact and not by uptake of secreted fibroblast enzyme. The process is dependent on time and on the number of fibroblasts used. Electron-microscopic examination of the co-culture of the two cell types reveals extensive region of intimate contact. Fibroblastic projections appear frequently in close apposition with lymphoblast invaginations; a diffuse micropinocytotic activity is evident only in fibroblastic cells.

Hepatitis B virus DNA in chronic HBsAg carriers: correlation with HBeAg/anti-HBe status, anti-HD and liver histology.

Hepatitis B virus DNA was determined in the sera of 198 chronic hepatitis B surface antigen (HBsAg) carriers by the spot hybridization technique. The results were correlated with hepatitis Be antigen (HBeAg) and antibody (anti-HBe), delta antibody (anti-HD) and liver histology. All subjects had a liver biopsy. The prevalence of HBV DNA was 63% in HBeAg-positive subjects and 8.8% in anti-HBe positives. HBV DNA was not found more frequently in chronic HBsAg carriers who had histological evidence of liver disease than in carriers without such evidence. Anti-HD was detected in 48.5% of subjects, with an increasing trend (p less than 0.001) according to the severity of liver disease. Among patients with more severe liver disease (CAH and cirrhosis), HBV DNA and HBeAg were detected less frequently in anti-HD-positive than in anti-HD-negative subjects (7% vs. 42.3%, p less than 0.001 and 7% vs. 34.4%, p less than 0.005, respectively). These findings indicate that HDV infection jointly affects both HBeAg status and HBV DNA.

Animal models for lysosomal storage diseases: a new case of feline mucopolysaccharidosis VI.

Two long-haired Siamese cats are reported with clinical manifestations of human mucopolysaccharidosis VI (Maroteaux-Lamy disease): facial dysmorphia, dysostosis multiplex, paralysis. Urine of the two affected animals contained a high concentration of glycosaminoglycans, as detected by the dimethylmethylene blue test. Qualitative analysis, performed by thin-layer chromatography of the cetylpyridinium chloride-precipitable material, showed dermatan sulphate. Excessive incorporation of [35S]sulphate in the intracellular mucopolysaccharide of cultured fibroblasts and deficiency of arylsulphatase B in such cells indicate that these cats are affected by Maroteaux-Lamy disease. They should thus be considered the first European case of feline mucopolysaccharidosis VI.

Interaction between HDV and HBV infection in HBsAg-chronic carriers.

We studied the interaction between HBV and HDV infection in 149 consecutive subjects with HBsAg positive chronic hepatitis and in 22 chronic HBsAg healthy carriers. Liver HBcAg was detected in 52 (30.4%) of the 171 subjects. Of these 52, 35 were HBV-DNA and HBeAg positive, 11 HBV-DNA positive only; two HBeAg positive only and four were negative for both HBeAg and HBV-DNA. None of the 119 HBcAg-negative subjects had detectable HBV-DNA in serum. HD-Ag in hepatocytes was detected in 31 of the 171 subjects (18%); it was detectable in none of the 22 HBsAg healthy carriers, in four of the 56 patients with chronic persistent hepatitis (7.2%), in six of the 24 patients with chronic lobular hepatitis (25%), in 16 of the 40 patients with chronic active hepatitis (40%) and in five of the 29 with cirrhosis (17%). A presence of anti-HD in serum in the absence of liver HD-Ag was found in 54 of the 171 subjects (32%). This condition was observed not only in patients with a progressive disease (37.7% of chronic active hepatitis or cirrhosis and 33% of chronic lobular hepatitis), but also in healthy carriers (36%) and in chronic persistent hepatitis patients (21.4%). Liver HBcAg was detected in 6.4% of the 31 HD-Ag-positive patients, in 12.9% of the 54 HD-Ag-negative/anti-HD positive, but in 50% of the 86 with no marker of HDV infection. HDV appears to inhibit HBV genome and such inhibition may persist even when anti-HD is the only HDV marker detectable.