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Biomedical subjects

A Azzi

Publications and source records attributed to A Azzi.

At least 19 recordsLinked to original sources
PubMed2000

Magnet used for protein crystallization: novel attempts to improve the crystal quality.

The accuracy of the structures of biological macromolecules determined by X-ray crystallography is of fundamental importance, both for the understanding of life processes and for medical applications. The resolution of the structure is thus critical, and is largely determined by the quality of single crystals. Here we report the results of applying a magnetic field and a magnetization force during growth of the snake muscle fructose-1,6-bisphosphatase and human estrogenic 17beta-hydroxysteroid dehydrogenase crystals. For both enzyme proteins, the quality of the crystals improved with repeated assay, and their data sets were collected at significantly higher resolutions. These results coincide with a mechanism involving the reduction of convection, due to both the hydrodynamics within a magnet and the partially reduced gravity induced by a magnetization force. The density difference between the crystal and solution becomes less significant, and the sedimentation speed of the crystals is also reduced in the presence of the magnetization force.

17-Hydroxysteroid Dehydrogenases↗
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

Effect of vitamin E on the development of atherosclerosis.

The development of atherosclerosis is a multifactorial process in which both elevated plasma cholesterol levels and proliferation of smooth muscle cells play a central role. Numerous studies have suggested the involvement of oxidative processes in the pathogenesis of atherosclerosis and especially of oxidised low density lipoproteins. Some epidemiological studies have shown an association between high dietary intake or high serum concentrations of vitamin E and lower rates of ischemic heart disease. Recently, the Cambridge Heart Antioxidant Study (CHAOS) reported strong protection by high vitamin E doses against the risk of fatal and non fatal myocardial infarction. Here we have shown that incubation of vascular smooth muscle cells in the presence of alpha-tocopherol resulted in inhibition of cell proliferation and protein kinase C activity. Since beta-tocopherol and probucol are not inhibitory, the effect of alpha-tocopherol is considered due to a non-oxidant mechanism. In order to understand the protective role of alpha-tocopherol against atherosclerosis in vivo the following rabbit studies were carried out. Atherosclerosis was induced by a vitamin E poor diet containing 2% cholesterol in a group of rabbit. The other groups had 2% cholesterol in the diet plus 50 mg/kg vitamin E i.m. or 1% probucol or 50 mg/kg vitamin E plus 1% probucol. After 4 weeks, aortas were removed and analysed by microscopy for atherosclerotic lesions. Samples of the media were analysed for protein kinase C activity. The aortas of cholesterol-fed rabbits showed typical atherosclerotic lesions, detected by microscopic examination, their media smooth muscle cells exhibited an increase in protein kinase C activity. Vitamin E fully prevented cholesterol induced atherosclerotic lesions and the induction of protein kinase C activity while probucol was not effective. These results show that the protective effect of vitamin E against hypercholesterolemic atherosclerosis is not produced by an other antioxidant such as probucol and, therefore, may not be linked to the antioxidant properties of this vitamin. The effects observed at the level of smooth muscle cells in vitro and ex-vivo suggests an involvement of signal transduction events in the protective effect of vitamin E against atherosclerosis.

Animals↗
PubMed2000

Vitamin E reduces the uptake of oxidized LDL by inhibiting CD36 scavenger receptor expression in cultured aortic smooth muscle cells.

BACKGROUND: Vitamin E is well known as an antioxidant, and numerous studies suggest that it has a preventive role in atherosclerosis, although the mechanism of action still remains unclear. METHODS AND RESULTS: The original aim of this study was to establish whether alpha-tocopherol (the most active form of vitamin E) acts at the earliest events on the cascade of atherosclerosis progression, that of oxidized LDL (oxLDL) uptake and foam-cell formation. We show here that the CD36 scavenger receptor (a specific receptor for oxLDL) is expressed in cultured human aortic smooth muscle cells (SMCs). Treatment of SMCs and HL-60 macrophages with alpha-tocopherol (50 micromol/L, a physiological concentration) downregulates CD36 expression by reducing its promoter activity. Furthermore, we find that alpha-tocopherol treatment of SMCs leads to a reduction of oxLDL uptake. CONCLUSIONS: This study indicates that CD36 is expressed in cultured human SMCs. In these cells, CD36 transports oxLDL into the cytosol. alpha-Tocopherol inhibits oxLDL uptake by a mechanism involving downregulation of CD36 mRNA and protein expression. Therefore, the beneficial effect of alpha-tocopherol against atherosclerosis can be explained, at least in part, by its effect of lowering the uptake of oxidized lipoproteins, with consequent reduction of foam cell formation.

Aorta↗
PubMed2000

Scavenger receptor regulation and atherosclerosis.

Atherosclerosis and its complications, such as coronary heart disease, heart infarction and stroke, are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. During atherosclerosis, uncontrolled cholesterol and lipid accumulation in macrophages and smooth muscle cells leads to foam cell formation and to the progression of the atherosclerotic plaque. This review will focus on foam cell formation within the atherosclerotic lesion, the involvement of the scavenger receptor genes in this process, and the possibility to interfere with scavenger receptor function to reduce the progression of atherosclerosis. To date, the regulatory mechanisms for the expression of scavenger receptor genes and their role in atherosclerosis are not well characterized. Knowledge on this subject could lead to a better understanding of the process, prevention and therapy of this disease.

Animals↗
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

Use of denaturing gradient gel electrophoresis for human polyomavirus JC sequence analysis.

Five genotypes of human polyomavirus JC (JCV) have been detected so far by nucleotide sequencing and by restriction fragment length polymorphism analysis. These genotypes are the result of geographically based virus evolution. However, interesting aspects of genotyping could involve both pathogenetic and diagnostic aspects. The product from amplification of JCV sequences, found in urine from 12 healthy individuals from different countries, have been analysed by denaturing gradient gel electrophoresis (DGGE) and by nucleotide sequencing. The aim of this study was to assess if the DGGE analysis could be used to study the variability of the JCV genome. The target sequence of this study was 233 bp long, within the gene coding for the VP1, known to contain several type-determining sites. Four DGGE patterns have been observed among our strains. Five strains, from African individuals, were of type 3 and exhibited the same electrophoretic pattern, clearly distinguishable from that of the type 1 strains detected in the urine from 6 European individuals. Five type 1 strains shared a similar DGGE pattern, slightly different from that of the sixth. A different pattern characterised as a type 2 strain was detected in the urine from a Peruvian individual. These results suggest that DGGE analysis could be used as a screening assay for choosing strains for nucleotide sequencing. The analysis of a fragment larger than the one used in this study could allow the identification of more types and subtypes.

Adolescent↗
PubMed2000

Sequential vidarabine infusion in the treatment of polyoma virus-associated acute haemorrhagic cystitis late after allogeneic bone marrow transplantation.

Late onset haemorrhagic cystitis (HC) occurs in 20-30% of allogeneic bone marrow transplant patients. Human polyomavirus BK (BKV) (or less frequently adenovirus) may be involved in the pathogenesis of viral HC and can represent a serious post-transplant complication. Diagnosis and treatment of viral HC can be difficult and has an uncertain outcome. We report the efficacy of sequential vidarabine in the treatment of a patient with severe BKV-associated HC, despite the delay in implementing therapy. Bone Marrow Transplantation (2000) 25, 319-320.

Acute Disease↗
PubMed2000

Scavenger receptors and modified lipoproteins: fatal attractions?

Lipoproteins modified by oxidation, glycation, alkylation, and nitration are generated by oxidative stress during inflammation, diabetes, and inadequate supply of dietary antioxidants. A family of genes, the scavenger receptors, recognizes and internalizes modified lipoproteins, making them susceptible to degradation. Clearance of modified lipoproteins by scavenger receptors occurs mainly in macrophages, dendritic cells, and Kupffer cells of the liver. However, scavenger receptor expression also occurs in other cells, such as endothelial cells, aortic smooth muscle cells, neuronal cells, and keratinocytes. Thus, the local clearance of oxidized low-density lipoprotein and the resolution of inflammatory processes may rely in part on the expression of scavenger receptors in "nonprofessional" phagocytes. Uptake of oxidized low-density lipoprotein, without an efficient machinery to degrade them and uncontrolled expression of scavenger receptors, may lead to cellular deregulation, apoptosis, and formation of foam cells. Diseases accompanied by oxidation of lipoproteins, such as atherosclerosis, Alzheimer disease, glomerulosclerosis, ataxia with vitamin E deficiency, and possibly age-dependent lipofuscin deposition, may share a common pathogenetic feature. This review will focus on foam cell formation, mainly within the atherosclerotic lesion, and the possible involvement of aberrant regulation of the scavenger receptor genes. To date, the regulatory mechanisms at the basis of scavenger receptor gene expression and their roles in atherosclerosis and other diseases are not well established. Knowledge on this subject could lead to a better understanding of the pathogenesis, prevention, and therapy of these diseases.

Animals↗
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↗
PubMed1999

Modulation of alpha-tropomyosin expression by alpha-tocopherol in rat vascular smooth muscle cells.

The effect of alpha-tocopherol (vitamin E) on gene expression in rat vascular smooth muscle cells was studied by the differential display technique. One gene out of about 1000 genes analyzed, identified as alpha-tropomyosin, showed an increased transcription level caused by alpha-tocopherol treatment. Northern and Western blot analysis revealed a time-dependent transient up-regulation of the amount of mRNA (peak between 2 and 3 h) and protein (peak at 5 h) in alpha-tocopherol-treated cells. No effect was observed in cells treated with beta-tocopherol.

Animals↗
PubMed1999

Signal transduction in rat vascular smooth muscle cells: control of osmotically induced aldose reductase expression by cell kinases and phosphatases.

We have studied the osmotically induced gene expression (measured as chloramphenicol acetyl transferase (CAT) reporter gene expression) in rat smooth muscle cell primary cultures (rSMC), under the control of osmotic response elements (ORE). It was found that osmotically induced gene expression is sensitive to signal transduction inhibitors and activators. In particular, protein kinase C inhibition by calphostin C prevented gene expression by osmotic response. On the other hand, receptor tyrosine kinase inhibition has been shown to produce an enhancement of gene expression. This suggests that tyrosine kinase receptor activation exerts an inhibitory action on ORE induced gene expression. Gene expression was also induced by treating cells with PD098059, a specific inhibitor of mitogen-activated protein kinase kinase. Moreover, the same inhibitors and activators have been shown to affect the hyperosmosis induced expression of aldose reductase gene.

Aldehyde Reductase↗
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

Age-dependent increase of collagenase expression can be reduced by alpha-tocopherol via protein kinase C inhibition.

Total protein kinase C (PKC) activity in human skin fibroblasts increases during in vivo aging as a function of the donor's age. During in vitro aging protein kinase C activity is also increased, as a function of cell passage number. Using PKC isoform specific antibodies, we demonstrate that the increase in total PKC activity is mainly due to the PKC a isoform. PKC alpha protein expression increased up to 8 fold during in vivo aging. Collagenase (MMP-1) gene transcription and protein expression also increased with age, concomitant with the increase in protein kinase C alpha. Furthermore, alpha-tocopherol, which inhibits protein kinase C activity, is able to diminish collagenase gene transcription without altering the level of its natural inhibitor, tissue inhibitor of metalloproteinase, TIMP-1. We propose that an aging program leads to increased protein kinase C alpha expression and activity. This event would induce collagenase overexpression followed by increased collagen degradation. Our in vitro experiments with skin fibroblasts suggest that alpha-tocopherol may protect against skin aging by decreasing the level of collagenase expression, which is induced by environmental insults and by aging.

Adult↗
PubMed1999

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17Beta-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41). To further study the transition state, two non-reactive ternary complexes, 17beta-HSD1-EM519-NADP+ and 17beta-HSD1-EM553-NADP+ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a = 155.59 A, b = 42.82 A, c = 121.15 A, beta = 128.5 degrees for 17beta-HSD1-EM519-NADP+, and a = 124.01 A, b = 45.16 A, c = 61.40 A, beta = 99.2 degrees for 17beta-HSD1-EM553-NADP+, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Human polyomavirus BK (BKV) load and haemorrhagic cystitis in bone marrow transplantation patients.

Several observations suggest an association between long-lasting haemorrhagic cystitis (HC) in bone marrow transplantation (BMT) recipients and human polyomavirus BK (BKV) reactivation, but no conclusive evidence has been obtained so far. The amount of BKV measured in the urine of BMT patients during an episode of HC was compared with that detected in the urine of BMT patients without HC and of immunocompetent individuals in order to better assess the association of BKV reactivation with HC. For this purpose a quantitative competitive PCR was developed. The application of this assay to clinical samples allowed us to distinguish asymptomatic reactivation both in healthy individuals and in immunocompromised patients from reactivation associated with HC, in almost all cases. Low levels, below the sensitivity of the quantitative assay, were shown in asymptomatic healthy individuals and in about 50% of immunocompromised patients. A significantly higher viral load than in the urine of asymptomatic immunocompromised patients was detected in the urine of patients with HC. These data strengthen the hypothesis that BKV reactivation can cause, together with other factors, the majority of late HC in BMT recipients as well as in patients treated for acute refractory lymphoblastic leukemia.

Adolescent↗