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A Stocker

Publications and source records attributed to A Stocker.

At least 19 recordsLinked to original sources
PubMed2001

Nonantioxidant functions of alpha-tocopherol in smooth muscle cells.

Most tocopherols and tocotrienols, with the exception of alpha-tocopherol, are not retained by humans. This suggests that alpha-tocopherol is recognized uniquely; therefore, it may exert an exclusive function. alpha-Tocopherol possesses distinct properties that are independent of its prooxidant, antioxidant or radical-scavenging ability. alpha-Tocopherol specifically inhibits protein kinase C, the growth of certain cells and the transcription of the CD36 and collagenase genes. Activation events have also been seen on the protein phosphatase 2A (PP(2)A) and on the expression of other genes (alpha-tropomyosin and connective tissue growth factor). Neither ss-tocopherol nor probucol possessed the same specialty functions as alpha-tocopherol. Recently, we isolated a new ubiquitous cytosolic alpha-tocopherol binding protein (TAP). Its motifs suggest that it is a member of the hydrophobic ligand-binding protein family (CRAL-TRIO). TAP may also be involved in the regulation of cellular alpha-tocopherol concentration and alpha-tocopherol-mediated signaling.

Cells, Cultured↗
PubMed2000

A novel human tocopherol-associated protein: cloning, in vitro expression, and characterization.

Vitamin E (alpha-tocopherol) is an essential dietary nutrient for humans and animals. The mechanisms involved in cellular regulation as well as in the preferential cellular and tissue accumulation of alpha-tocopherol are not yet well established. We previously reported (Stocker, A., Zimmer, S., Spycher, S. E., and Azzi, A. (1999) IUBMB Life 48, 49-55) the identification of a novel 46-kDa tocopherol-associated protein (TAP) in the cytosol of bovine liver. Here, we describe the identification, the molecular cloning into Escherichia coli, and the in vitro expression of the human homologue of bovine TAP, hTAP. This protein appears to belong to a family of hydrophobic ligand binding proteins, which have the CRAL (cis-retinal binding motif) sequence in common. By using a biotinylated alpha-tocopherol derivative and the IASys resonant mirror biosensor, the purified recombinant protein was shown to bind tocopherol at a specific binding site with K(d) 4.6 x 10(-7) m. Northern analyses showed that hTAP mRNA has a size of approximately 2800 base pairs and is ubiquitously expressed. The highest amounts of hTAP message are found in liver, brain, and prostate. In conclusion, hTAP has sequence homology to proteins containing the CRAL_TRIO structural motif. TAP binds to alpha-tocopherol and biotinylated tocopherol, suggesting the existence of a hydrophobic pocket, possibly analogous to that of SEC14.

Amino Acid Motifs↗
PubMed2000

Vitamin E: non-antioxidant roles.

Vitamin E was originally considered a dietary factor of animal nutrition especially important for normal reproduction. The significance of vitamin E has been subsequently proven as a radical chain breaking antioxidant that can protect the integrity of tissues and play an important role in life processes. More recently alpha-tocopherol has been found to possess functions that are independent of its antioxidant/radical scavenging ability. Absorption in the body is alpha-tocopherol selective and other tocopherols are not absorbed or are absorbed to a lesser extent. Furthermore, pro-oxidant effects have been attributed to tocopherols as well as an anti-nitrating action. Non-antioxidant and non-pro-oxidant molecular mechanisms of tocopherols have been also described that are produced by alpha-tocopherol and not by beta-tocopherol. alpha-Tocopherol specific inhibitory effects have been seen on protein kinase C, on the growth of certain cells and on the transcription of some genes (CD36, and collagenase). Activation events have been seen on the protein phosphatase PP2A and on the expression of other genes (alpha-tropomyosin and Connective Tissue Growth Factor). Non-antioxidant molecular mechanisms have been also described for gamma-tocopherol, delta-tocopherol and tocotrienols.

Antioxidants↗
PubMed2000

Tocopherol-binding proteins: their function and physiological significance.

The present review is a continuation of earlier essays on the uptake mechanisms and the biological function of vitamin E. There are eight naturally occurring homologues of vitamin E, which differ in their structure and in biological activity in vivo and in vitro. Various studies have suggested that after normal gastrointestinal absorption of dietary vitamin E specific mechanisms favor the preferential accumulation of one of its homologues, alpha-tocopherol, in the human body. This process is thought to be mediated in part by the alpha-tocopherol transfer protein (alpha-TTP) in the liver cytoplasm. The mechanism and pathway by which alpha-TTP specifically incorporates alpha-tocopherol into plasma lipoproteins is not yet fully understood. Because alpha-tocopherol is widely distributed in tissues in various concentrations but alpha-TTP resides only in liver, its role as intracellular carrier of alpha-tocopherol seems unlikely. However, recent data indicate that a system of alpha-tocopherol-binding proteins is involved in these processes that favor the localization of alpha-tocopherol at the sites where it is required. The current status of the evidence for the regulation of alpha-tocopherol levels and their impact on cellular signaling is discussed.

Absorption↗
PubMed2000

Specific cellular responses to alpha-tocopherol.

In the last 10 years precise cellular functions of alpha-tocopherol, some of which are independent of its antioxidant/radical-scavenging ability, have been revealed. Absorption of alpha-tocopherol from the gut is a selective process. Other tocopherols are not absorbed or are absorbed to a lesser extent. At the post-translational level, alpha-tocopherol inhibits protein kinase C and 5-lipoxygenase and activates protein phosphatase 2A and diacylglycerol kinase. Some genes [platelet glycoprotein IV/thrombospondin receptor/class B scavenger receptor (CD36), alpha-tocopherol transfer protein (alpha-TTP), alpha-tropomyosin, connective tissue growth factor and collagenase] are affected by alpha-tocopherol at the transcriptional level. alpha-Tocopherol also inhibits cell proliferation, platelet aggregation, monocyte adhesion and the oxygen burst in neutrophils. Other antioxidants, such as beta-tocopherol and probucol, do not mimic these effects, suggesting a nonantioxidant, alpha-tocopherol-specific molecular mechanism.

Animals↗
PubMed2000

Differential regulation of activation-induced cell death in individual human T cell clones.

BACKGROUND: Restimulation of T lymphocytes via the TCR/CD3 complex can result in CD95/CD95L-dependent activation-induced cell death (AICD). Although the correlation of AICD sensitivity to the T helper 1 phenotype was confirmed in different studies, the underlying mechanism is still debated. Thus, it has been suggested that in Th2 cells, AICD resistance is controlled by a TCR-induced upregulation of the CD95-associated inhibitory phosphatase, FAP-1. We and others demonstrated that AICD resistance is associated with a reduced surface expression of CD95L upon restimulation. METHODS: Utilizing RT-PCR, Western blotting and flow cytometry, we analyzed time-dependent changes in levels of CD95L mRNA, cytosolic protein and surface expression in five long-term human T cell clones and polarized helper populations. RESULTS: We confirm that the inducible CD95L surface expression is lower or absent in all tested AICD-resistant clones as compared to sensitive cells. It is of interest that striking differences with respect to the activation-dependent inducibility of CD95L mRNA expression in individual resistant clones were observed. In addition, alterations in the expression of the inhibitory phosphatase FAP-1 or TCR-dependent changes in CD95 sensitivity in AICD-resistant clones could be ruled out as a mechanism for AICD resistance of human T cell clones. CONCLUSIONS: (1) The data presented strongly support the previous notion that AICD resistance of human T cell clones is mainly regulated by a differential expression of CD95L. (2) Differential expression of CD95L on individual resistant clones results from a lack of mRNA induction in one set and from a markedly decreased surface expression of translated protein in another set of clones.

Antibodies, Monoclonal↗
PubMed1999

Vitamin E mediated response of smooth muscle cell to oxidant stress.

Oxidant stress is associated with diminution of antioxidant molecules, such as alpha-tocopherol. Alpha-tocopherol specifically decreases, in a concentration dependent way, the proliferation of vascular smooth muscle cells. At the same concentrations (10-50 microM) it induces inhibition of protein kinase C (PKC) activity. The latter event is not due to a decrease in PKC level or to alpha-tocopherol binding to PKC, but it results from increase of protein phosphatase 2A1 activity. In vitro data, as well as at a cellular level, demonstrates that protein phosphatase 2A1 is activated, in its trimeric structure--but not as a dimer by alpha-tocopherol. This activation is followed by PKC-alpha dephosphorylation. The activation of protein phosphatase 2A1 and deactivation of PKC-alpha affect the AP1 transcription factor, resulting in a change in the composition and the binding of this factor to DNA. By transfecting smooth muscle cell with a construct containing three TRE (TPA responsive elements), the promoter thymidine kinase and the reporter gene chloramphenicol-acetyl-transferase a modulation of gene expression by alpha-tocopherol is observed. Beta-tocopherol does not cause any of the responses observed with alpha-tocopherol and R,R,R-alpha-tocopherol is twice as potent as all-rac-alpha-tocopherol. When added together, beta-tocopherol prevents the effects of alpha-tocopherol indicating that the mechanism involved is not related to the radical-scavenging properties of these two molecules, which are essentially equal. By differential display analysis it has been found that several genes of smooth muscle cells are differentially transcribed in the presence of alpha-tocopherol but not beta-tocopherol. In particular, the gene of alpha-tropomyosin shows a transient enhancement of transcription as a function of the cell cycle time. Alpha-tropomyosin translation is also increased by alpha-tocopherol and not by beta-tocopherol. Because no changes of mRNA stability can be observed in the presence of alpha-tocopherol, the data supports the conclusion of a transcriptional control exerted by alpha-tocopherol on alpha-tropomyosin. Generally, the data strongly suggests the existence of a ligand/receptor type of mechanism at the basis of alpha-tocopherol action. It is concluded that an oxidative stress-induced diminution of alpha-tocopherol in smooth muscle cell activates a reaction cascade leading to changes in gene expression and increase in cell proliferation by a non-antioxidant mechanism.

Animals↗
PubMed1999

Covalent flavinylation of L-aspartate oxidase from Escherichia coli using N6-(6-carboxyhexyl)-FAD succinimidoester.

L-Aspartate oxidase is a flavoprotein catalyzing the first step in the de novo biosynthesis of pyridine nucleotides in E. coli. Binding of FAD to L-aspartate oxidase is relatively weak (K(d) 6.7 x 10(-7) M), resulting in partial loss of the coenzyme under many experimental conditions. Only the three-dimensional structure of the apo-enzyme has been obtained so far. In order to probe the flavin-binding site of the enzyme, apo-L-aspartate oxidase has been reacted with N6-(6-carboxyhexyl)-FAD succinimidoester. The structural characterization of the resulting N6-(6-carbamoylxyhexyl)FAD-L-aspartate oxidase shows the covalent incorporation of 1 FAD-analog/monomer. Residue Lys38 was identified as the target of the covalent modification. N6-(6-carbamoylxyhexyl)-FAD-L-aspartate oxidase shows only 2% catalytic activity as compared to the native enzyme. Comparison of some properties of the flavinylated and native enzymes suggests that, although the flavin is covalently bound to the former in the region predicted from molecular modeling studies, the microenvironment around the isoallossazine is different in the two forms.

Amino Acid Oxidoreductases↗
PubMed1999

Identification of a novel cytosolic tocopherol-binding protein: structure, specificity, and tissue distribution.

Alpha-tocopherol plays an important role as a lipid-soluble antioxidant. It is present in all major mammalian cell types and shows tissue-specific distribution. This suggests the presence of specific proteins involved in intracellular distribution or metabolism of alpha-tocopherol. A diminution of tocopherol plasma concentrations contributes to the development of diseases such as vitamin E deficiency (AVED), atherosclerosis, and prostate cancer. Further evidence has been obtained for the existence of sites in cellular metabolism and signal transduction where alpha-tocopherol potentially plays a regulatory role. A signal transduction modulation specific for alpha-tocopherol has been described in several model systems. Using radioactively labeled alpha-tocopherol as tracer, we have isolated a new alpha-tocopherol-associated protein (TAP) from bovine liver. This protein has a molecular mass of 46 kDa and an isoelectric point of 8.1. From its partial amino acid sequence, a human gene has been identified with high homology to the newly described protein. Sequence analysis has established that the new TAP has structural motifs suggesting its belonging to a family of hydrophobic ligand-binding proteins (RALBP, CRALBP, alpha-TTP, SEC 14, PTN 9, RSEC 45). Human TAP has been cloned into Escherichia coli, and its tissue-specific expression has been assessed by Northern blot analysis.

Amino Acid Sequence↗
PubMed1997

Signalling functions of alpha-tocopherol in smooth muscle cells.

alpha-Tocopherol but not beta-tocopherol, activates protein phosphatase 2A, decreases protein kinase C activity and attenuates smooth muscle cell proliferation at physiological concentrations. beta-Tocopherol prevents the effects of alpha-tocopherol. Inhibition of protein kinase C alpha, but not of the other isoforms, by the inhibitor Gö6976 prevents the effect of alpha-tocopherol. Protein kinase C alpha, immunoprecipitated from alpha-tocopherol treated cells, is less phosphorylated and inactive. It is proposed that the specific activation of protein phosphatase 2A by alpha-tocopherol results in dephosphorylation and inactivation of protein kinase C alpha. Finally, this cascade of events leads to smooth muscle cell proliferation inhibition.

Animals↗
PubMed1996

Synthesis, characterization and preliminary crystallographic data of N6-(6-carbamoylhexyl)-FAD-D-amino-acid oxidase from pig kidney, a semi-synthetic oxidase.

The FAD analogue, N6-(6-carboxyhexyl)-FAD, carrying a hexanoic acid residue at the N6 position of the adenine moiety was synthesized. A new semi-synthetic oxidase, N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase, was prepared by reacting the succinimido ester of N6-(6-carboxyhexyl)-FAD with apo-D-amino-acid oxidase from pig kidney in the presence of benzoate. Reaction conditions and methods have been developed for preparing pure semi-synthetic and fully active N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase that contains 1 covalently bound FAD analogue/subunit, as verified by redialysis, ultraviolet spectrophotometry, electrospray ionization (ESI)-MS and peptide mapping. Presumably, the N6-(6-carbamoylhexyl)-FAD moiety of this semi-synthetic D-amino-acid oxidase (DAAO), selectively bound to Lys163, has a structurally similar position to that of the non-covalently bound FAD of the native holoenzyme, since both DAAO forms show very similar kinetic properties (semi-synthetic DAAO, Vmax(app) = 17.7 mumol min-1 mg-1; KM(app) = 4.5 mM; native holo-DAAO, Vmax = 12.2 mumol min-1 mg-1; KM = 1.8 mM). Compared with the native holo-D-amino acid oxidase. this new semi-synthetic N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase is a considerably more stable enzyme that shows meso-thermostability and withstands inactivation on dilution. Probably, the lack of dissociation of FAD and, consequently, the absence of the instable apoenzyme are responsible for these phenomena. Preliminary investigations resulted in finding convenient and reproducible crystallization conditions for N6-(6-carbamoylhexyl)-FAD-D-amino acid oxidase. The single crystals, obtained by the sitting-drop method using ammonium sulfate as precipitant, belong to the tetragonal space group I422 with cell dimensions a = 16.3 nm, c = 13.6 nm. The crystals diffract to 0.3-nm resolution, with two molecules being present in the asymmetric unit, demonstrating the two-subunit quarternary structure of this semi-synthetic D-amino-acid oxidase.

Animals↗
PubMed1996

Purification and some properties of a novel microbial lactate oxidase.

Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron microscopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45 degrees C. The addition of glycerol and dextran 500,000 to the enzyme sample enhanced storage stability.

Amino Acid Sequence↗
PubMed1996

The substrate specificity of tocopherol cyclase.

The substrate specificity of the enzyme tocopherol cyclase from the blue-green algae Anabaena variabilis (Cyanobacteria) was investigated with 11 substrate analogues revealing the significance of three major recognition sites: (i) the OH group at C(1) of the hydroquinone, (ii) the (E) configuration of the double bond, and (iii) the length of the lipophilic side chain. Experiments with two affinity matrices suggest that substrates approach the enzyme's active site with the hydrophobic tail.

Chemical Phenomena↗
PubMed1995

Improving enzyme-electrode contacts by redox modification of cofactors.

Efficient electron transfer of redox proteins to and from their environment is essential for the use of such proteins in biotechnological applications such as amperometric biosensors and photosynthetic biocatalysts. But most redox enzymes lack pathways that can transport an electron from their embedded redox site to an electrode or a diffusing photoexcited species. Electrical communication between redox proteins and electrode surfaces has been improved by aligning proteins on chemically modified electrodes, by attaching electron-transporting groups and by immobilizing proteins in polymer matrices tethered by redox groups. Generally these methods involve contacting the enzymes at random with electron relay units. Here we report an approach that allows site-specific positioning of electron-mediating units in redox proteins. We strip glucose oxidase of its flavin adenine dinucleotide (FAD) cofactors, modify the latter with redox-active ferrocene-containing groups, and then reconstitute the apoprotein with these modified cofactors. In this way, electrical contact between an electrode and the resulting enzyme in solution is greatly enhanced in a controlled and reproducible way.

Apoproteins↗
PubMed1995

High-flow, small-hole arteriovenous fistulas: treatment with electrodetachable coils.

We present one case of carotid-cavernous fistula caused by percutaneous treatment of trigeminal neuralgia and one case of vertebrovertebral fistula caused by percutaneous internal jugular vein cannulation. Each fistula had a small arteriovenous communication that prevented the use of detachable balloons. Endovascular transarterial treatment of these two iatrogenic fistulas with electrically detachable platinum coils was performed. Both fistulas were occluded with preservation of the parent artery, and the patients have fully recovered.

Aged↗
PubMed1993

Active neurocysticercosis, parenchymal and extraparenchymal: a study of 38 patients.

In this retrospective study we have analysed a series of 38 patients seen from 1983 to 1992 (mean follow-up, 4.5 years) with active neurocysticercosis (NCC), 23 (60.5%) with parenchymal and 15 (39.5%) with extraparenchymal NCC. Classification into these two forms of NCC was based on computed tomography and magnetic resonance imaging criteria. The enzyme-linked immunosorbent assay performed in cerebrospinal fluid, for anti-Taenia solium antibodies, was positive in 18 of 23 (78%) cases. Epilepsy and/or intracranial hypertension were the most common clinical presentation (92%). Twenty-three (60.5%) of 38 patients were treated with praziquantel and/or albendazole. In parenchymal NCC, the efficacy of medical therapy was complete in 13 of 16 (81%) and partial in 3 of 16 (19%) patients. In contrast, in all cases of extraparenchymal NCC treated with cysticidal drugs the results were disappointing. A ventriculoperitoneal shunt was performed in 9 of 13 patients with extraparenchymal NCC and hydrocephalus. Severe complications, including two deaths, associated with the natural evolution of the disease or with surgery, occurred only in extraparenchymal NCC. Therefore, we confirm the existence of the two forms of active NCC, parenchymal and extraparenchymal, which are strikingly different in clinical presentation, medical therapy response, complications, morbidity and mortality.

Adolescent↗