Biomedical subjects

Jeffrey Atkinson

Publications and source records attributed to Jeffrey Atkinson.

At least 19 recordsLinked to original sources

PubMed2007

Animal models of arterial stiffness.

Animal models of large artery wall stiffness fall into two categories: firstly those that slowly develop multifactorial vascular dysfunction spontaneously, such as the ageing rat. The second type of model consists of those in which a specific pathology is induced by surgical, chemical, or genetic means. Such models are based on a short-term, highly traumatic insult to the arterial wall of a young animal and its acute reaction to such insult. This is very different from the human situation in which changes in wall stiffness arise from the long-term accumulation of relatively minor episodes of vascular insult in the vulnerable elderly.

Animals↗

PubMed2006

Biochemical consequences of heritable mutations in the alpha-tocopherol transfer protein.

Tocopherol transfer protein (TTP) regulates vitamin E status by facilitating the secretion of tocopherol from liver to circulating lipoproteins. Heritable mutations in the ttpA gene, encoding for TTP, result in ataxia with vitamin E deficiency (AVED) syndrome, typified by low vitamin E levels and a plethora of neurological disorders. The molecular mechanisms by which TTP facilitates tocopherol secretion are presently unknown. We recently showed that vitamin E is taken up by hepatocytes through an endocytic process and that, shortly following uptake, the vitamin is found primarily in lysosomes. We showed further that TTP is localized to late endocytic vesicles and that it facilitates the intracellular trafficking of tocopherol from lysosomes to the plasma membrane. To gain insight into the molecular mechanisms that underlie TTP actions, we studied the physiological impact of three naturally occurring heritable mutations in the ttpA gene (the R59W, R221W, and A120T substitutions). We found that these mutations impair the ability of TTP to facilitate the secretion of vitamin E from cells. Furthermore, the degree of impairment corresponded to the severity of the AVED pathology associated with each mutation. In cells that express mutated TTP proteins, vitamin E did not traffic to the plasma membrane and remained "trapped" in lysosomes. In addition, we observed that substitution mutations that cause the AVED syndrome impart a marked instability on the TTP protein. These observations suggest that the physiological role of TTP is anchored in its ability to direct vitamin E trafficking from the endocytic compartment to transport vesicles that deliver the vitamin to the site of secretion at the plasma membrane.

Ataxia↗

PubMed2006

Ventricular-arterial coupling in a rat model of reduced arterial compliance provoked by hypervitaminosis D and nicotine.

The vitamin D(3) and nicotine (VDN) model is one of isolated systolic hypertension (ISH) in which arterial calcification raises arterial stiffness and vascular impedance. The effects of VDN treatment on arterial and cardiac hemodynamics have been investigated; however, a complete analysis of ventricular-arterial interaction is lacking. Wistar rats were treated with VDN (VDN group, n = 9), and a control group (n = 10) was included without the VDN. At week 8, invasive indexes of cardiac function were obtained using a conductance catheter. Simultaneously, aortic pressure and flow were measured to derive vascular impedance and characterize ventricular-vascular interaction. VDN caused significant increases in systolic (138 +/- 6 vs. 116 +/- 13 mmHg, P < 0.01) and pulse (42 +/- 10 vs. 26 +/- 4 mmHg, P < 0.01) pressures with respect to control. Total arterial compliance decreased (0.12 +/- 0.08 vs. 0.21 +/- 0.04 ml/mmHg in control, P < 0.05), and pulse wave velocity increased significantly (8.8 +/- 2.5 vs. 5.1 +/- 2.0 m/s in control, P < 0.05). The arterial elastance and end-systolic elastance rose significantly in the VDN group (P < 0.05). Wave reflection was augmented in the VDN group, as reflected by the increase in the wave reflection coefficient (0.63 +/- 0.06 vs. 0.52 +/- 0.05 in control, P < 0.05) and the amplitude of the reflected pressure wave (13.3 +/- 3.1 vs. 8.4 +/- 1.0 mmHg in control, P < 0.05). We studied ventricular-arterial coupling in a VDN-induced rat model of reduced arterial compliance. The VDN treatment led to development of ISH and provoked alterations in cardiac function, arterial impedance, arterial function, and ventricular-arterial interaction, which in many aspects are similar to effects of an aged and stiffened arterial tree.

Animals↗

PubMed2006

Effects of dexamethasone and L-canavanine on the intracellular calcium-contraction relation of the rat tail artery during septic shock.

The intracellular mechanism by which sepsis lowers vascular reactivity and the subsequent reversal by dexamethasone or nitric oxide synthase (NOS) inhibitors remain unclear. We measured the sensitivity of contraction of the rat tail artery to intracellular Ca2+ in a model of polymicrobial septic shock. At 22 h after cecal ligation and puncture (CLP), rats were treated with an anti-inflammatory glucocorticoid (dexamethasone, 1 mg/kg ip), an inducible NOS inhibitor (L-canavanine, 100 mg/kg ip), or saline. At 24 h after CLP, endothelium-denuded, perfused segments of tail artery were loaded with the intracellular Ca2+-sensitive dye fura 2 in vitro. Intracellular Ca2+ concentration and perfusion pressure were measured simultaneously. The rightward shift of the perfusion pressure-intracellular Ca2+ mobilization curve after norepinephrine stimulation subsequent to CLP indicates decreased intracellular Ca2+ sensitivity of contraction. The relation was restored by dexamethasone (which also restored in vivo blood pressure and flow), but not by L-canavanine (which restored perfusion pressure by further mobilization of intracellular Ca2+). We conclude that CLP lowers vasomotion by lowering intracellular Ca2+ sensitivity, which can be restored with glucocorticoid treatment. The involvement of inducible NOS does not solely account for the sepsis-induced reduction in Ca2+ sensitivity of contraction.

Animals↗

PubMed2006

Preparation of fluorescent tocopherols for use in protein binding and localization with the alpha-tocopherol transfer protein.

Sixteen fluorescent analogues of the lipid-soluble antioxidant vitamin alpha-tocopherol were prepared incorporating fluorophores at the terminus of omega-functionalized 2-n-alkyl-substituted chromanols (1a-d and 4a-d) that match the methylation pattern of alpha-tocopherol, the most biologically active form of vitamin E. The fluorophores used include 9-anthroyloxy (AO), 7-nitrobenz-2-oxa-1,3-diazole (NBD), N-methyl anthranilamide (NMA), and dansyl (DAN). The compounds were designed to function as fluorescent reporter ligands for protein-binding and lipid transfer assays. The fluorophores were chosen to maximize the fluorescence changes observed upon moving from an aqueous environment (low fluorescence intensity) to an hydrophobic environment such as a protein's binding site (high fluorescence intensity). Compounds 9d (anthroyloxy) and 10d (nitrobenzoxadiazole), having a C9-carbon chain between the chromanol and the fluorophore, were shown to bind specifically and reversibly to recombinant human tocopherol transfer protein (alpha-TTP) with dissociation constants of approximately 280 and 60 nM, respectively, as compared to 25 nM for the natural ligand 2R,4'R,8'R-alpha-tocopherol. Thus, compounds have been prepared that allow the investigation of the rate of alpha-TTP-mediated inter-membrane transfer of alpha-tocopherol and to investigate the mechanism of alpha-TTP function at membranes of different composition.

Binding, Competitive↗

PubMed2006

The pre-ischaemic neuroprotective effects of the polyamine analogues BU43b and BU36b in permanent and transient focal cerebral ischaemia models in mice.

The present study investigated the neuroprotective potential of two novel polyamine analogues, BU43b and BU36b, when administered 30 min prior to cerebral ischaemia. Neuroprotection in a permanent and a transient focal cerebral ischaemia mouse model (induced by intraluminal middle cerebral artery occlusion (MCAO)) was investigated using a range of histological and behavioural assessments. In the permanent ischaemia model, BU43b reduced oedema and showed a trend towards reduction in %HLV (percentage hemisphere lesion volume) when administered at a dose of 30 mg/kg i.p. Following transient ischaemia, treatment with BU43b decreased the %HLV and reduced oedema when administered at 30 mg/kg. BU43b also improved the locomotor activity (LMA) in MCAO mice at both 20 mg/kg and 30 mg/kg doses. BU36b was less effective than BU43b in both the permanent and the transient models, with its most pronounced effect being a trend towards reduction in oedema in both models. These results demonstrate that BU43b administered 30 min before ischaemia provided a good level of neuroprotection in the two models of cerebral ischaemia used and may have potential as a neuroprotective treatment for stroke.

Animals↗

PubMed2006

Utility of a fluorescent vitamin E analogue as a probe for tocopherol transfer protein activity.

The tocopherol transfer protein (TTP) is a member of the CRAL-TRIO family of lipid binding proteins that facilitates vitamin E transfer between membrane vesicles in vitro. In cultured hepatocytes, TTP enhances the secretion of tocopherol to the media; presumably, tocopherol transfer is at the basis of this biological activity. The mechanism underlying ligand transfer by TTP is presently unknown, and available tools for monitoring this activity suffer from complicated assay procedure and poor sensitivity. We report the characterization of a fluorescent vitamin E analogue, (R)-2,5,7,8-tetramethylchroman-2-[9-(7-nitrobenz[1,2,5]oxadiazol-4-ylamino)nonyl]chroman-6-ol (NBD-TOH), as a sensitive and convenient probe for the ligand binding and transfer activities of TTP. Upon binding to TTP, NBD-TOH fluorescence is blue shifted, and its intensity is greatly enhanced. We used these properties to accurately determine the affinity of NBD-TOH to TTP. The analogue binds to TTP reversibly and with high affinity (K(d) = 8.5 +/- 6 nM). We determined the affinity of NBD-TOH to a TTP protein in which lysine 59 is replaced with a tryptophan. When occurring in humans, this heritable mutation causes the ataxia with vitamin E deficiency (AVED) disorder. We find that the affinity of NBD-TOH to this mutant TTP is greatly diminished (K(d) = 71 +/- 19 nM). NBD-TOH functioned as a sensitive fluorophore in fluorescent resonance energy transfer (FRET) experiments. Using the fluorescent lipids TRITC-DHPE or Marina Blue-DHPE as a donor or an acceptor for NBD-TOH fluorescence, we obtained high-resolution kinetic data for tocopherol movement out of lipid bilayers, a key step in the TTP-facilitated ligand transfer reaction.

4-Chloro-7-nitrobenzofurazan↗

PubMed2006

Metalloproteinase-9 in circulating monocytes in pulmonary hypertension.

The role of matrix metalloproteinases (MMPs) in pulmonary hypertension (PH) is complex as MMPs are involved in both the vascular and cardiac remodelling associated with PH. To gain insight into this problem, monocytes were isolated from pulmonary arterial blood in patients suffering from PH, related to chronic obstructive pulmonary disease (n = 6), chronic pulmonary thromboembolism (n = 3) or pulmonary arterial hypertension (n = 8). The severity of PH was associated with decreases in cardiac index (CI) and mixed venous blood oxygen saturation (SO(2)), and an increase in right atrial pressure (). Monocyte pro-MMP-9 content (zymography) was positively correlated with SO(2) (r = 0.73, P < 0.05) and CI (r = 0.66, P < 0.05), and negatively with (r = 0.54, P < 0.05); there was no significant correlation with pulmonary vascular resistance. In conclusion, the pro-MMP-9 content of circulating monocytes was lower in the more severe forms of PH which showed heart failure suggesting that such MMP enzymatic activity reflects heart failure following pulmonary vascular and myocardial remodelling in PH.

Biomarkers↗

PubMed2005

Constrictor and dilator effects of angiotensin II on cerebral arterioles.

BACKGROUND AND PURPOSE: In light of the equivocal data on the cerebral vasoconstrictor and vasodilator actions of angiotensin II (Ang II) and the potential clinical importance of this, we investigated the effects of Ang II on rat pial arterioles. METHODS: We determined the effect of Ang I (3.10(-6) mol/L) in the absence and presence of the converting enzyme inhibitor, captopril (10(-5) mol/L) in cerebral arterioles of male Wistar rats (open-skull preparation), and those of Ang II (3.10(-12) to 3.10(-6) mol/L) in the absence and presence of the Ang II receptor (AT1) antagonist, telmisartan (10(-5) mol/L) or the AT2 antagonist, PD123319 (10(-5) mol/L). We examined the effect of PD123319 (10(-5) mol/L) and the Ca2+-activated K+ (BKCa) channel blocker, tetraethylammonium (10(-4) mol/L) on the Ang II responses in the presence of telmisartan (10(-5) mol/L). RESULTS: Ang II-induced dose-dependent constriction with a maximum decrease of -20.1+/-1.0% at 10(-6) mol/L. Captopril significantly decreased Ang I-induced vasoconstriction (-4.0+/-0.9 versus -21.3+/-2.5%; n=4). Telmisartan reversed Ang II-induced vasoconstriction (9.5+/-2.5 versus -20.1+/-1% at 10(-6) mol/L; n=5). PD123319 significantly increased Ang II-induced vasoconstriction (-12.9+/-0.8 versus -10.2+/-0.4% at 10(-6) mol/L; n=5). PD123319 abolished (-2.6+/-0.7 versus 9.3+/-1.1% at 10(-6) mol/L; n=5) whereas tetraethylammonium reversed (-12.1+/-1.6 versus 9.9+/-1.0% at 10(-6) mol/L; n=4) Ang II-induced vasodilatation in the presence of telmisartan. CONCLUSIONS: Angiotensin is converted locally into Ang II; the overall effect of Ang II is vasoconstrictor following stimulation of the AT1 receptor, but a vasodilator response can be evoked following stimulation of the AT2 receptor and activation of BKCa.

Angiotensin II↗

PubMed2005

Intracellular trafficking of vitamin E in hepatocytes: the role of tocopherol transfer protein.

The term vitamin E denotes a family of tocopherols and tocotrienols, plant lipids that are essential for vertebrate fertility and health. The principal form of vitamin E found in humans, RRR-alpha-tocopherol (TOH), is thought to protect cells by virtue of its ability to quench free radicals, and functions as the main lipid-soluble antioxidant. Regulation of vitamin E homeostasis occurs in the liver, where TOH is selectively retained while other forms of vitamin E are degraded. Through the action of tocopherol transfer protein (TTP), TOH is then secreted from the liver into circulating lipoproteins that deliver the vitamin to target tissues. Presently, very little is known regarding the intracellular transport of vitamin E. We utilized biochemical, pharmacological, and microscopic approaches to study this process in cultured hepatocytes. We observe that tocopherol-HDL complexes are efficiently internalized through scavenger receptor class B type I. Once internalized, tocopherol arrives within approximately 30 min at intracellular vesicular organelles, where it co-localizes with TTP, and with a marker of the lysosomal compartment (LAMP1), before being transported to the plasma membrane in a TTP-dependent manner. We further show that intracellular processing of tocopherol involves a functional interaction between TTP and an ABC-type transporter.

ATP Binding Cassette Transporter 1↗

PubMed2005

Pioglitazone improves aortic wall elasticity in a rat model of elastocalcinotic arteriosclerosis.

Specific treatment of age-related aortic wall arteriosclerosis and stiffening is lacking. Because ligands for peroxisome proliferator-activated receptor gamma have beneficial effects on the arterial wall in atherosclerosis, via an antiinflammatory mechanism, we investigated whether long-term pioglitazone (Pio) treatment protects against another form of vascular wall disease, arteriosclerosis. We evaluated, in a rat model of elastocalcinotic arteriosclerosis (hypervitaminosis D and nicotine [VDN]), whether Pio (3 mg . kg(-1) per day for 1.5 month PO) attenuated arteriosclerosis and its consequences: aortic wall rigidity, increased aortic pulse pressure, and left ventricular hypertrophy. In VDN rats, medial calcification was associated with monocyte/macrophage infiltration and induction of tumor necrosis factor alpha and interleukin 1beta. Pio increased nuclear peroxisome proliferator-activated receptor gamma immunostaining in the aortic wall, decreased tumor necrosis factor alpha (P <0.05 versus VDN Pio-), tended to decrease interleukin 1beta mRNA expression (P =0.08 versus VDN Pio-), blunted aortic wall calcification (271+/-69, P <0.05 versus VDN Pio- 562+/-87 micromol . g(-1) dry weight) and prevented fragmentation of elastic fibers (segments per 10,000 microm2: 8.4+/-0.3; P <0.05 versus VDN Pio- 10.5+/-0.6). Pio reduced aortic wall stiffness (elastic modulus/wall stress: 4.8+/-0.6; P <0.05 versus VDN Pio- 10.0+/-1.6), aortic pulse pressure (30+/-2 mm Hg; P <0.05 versus VDN Pio- 39+/-4) and left ventricular hypertrophy (1.58+/-0.05 g . kg(-1); P <0.05 versus VDN Pio- 1.76+/-0.06). In conclusion, long-term Pio treatment attenuates aortic wall elastocalcinosis and, thus, lowers aortic wall stiffness, aortic pulse pressure, and left ventricular hypertrophy.

Animals↗

PubMed2005

Captopril improves cerebrovascular structure and function in old hypertensive rats.

We examined the effects of an angiotensin-converting enzyme inhibitor (ACEI), captopril, on cerebral arterioles in young and old spontaneously hypertensive rats (SHR). Animals were anesthetized with sodium pentobarbitone (60 mg kg(-1) day(-1)). We measured cerebral blood flow (CBF, arbitrary units) and cerebral arteriolar internal diameter (ID, mum) prior to and during stepwise hypotension (SH) in 6- (WKY-6) and 15-month-old (WKY-15) Wistar Kyoto rats and in age-matched SHR that were untreated (SHR-6 and SHR-15) or treated for 3 months with captopril (SHR-6C, 105+/-2 mg kg(-1) day(-1) and SHR-15C, 94+/-1 mg kg(-1) day(-1)). ID and cross-sectional area of the vessel wall (CSA) were measured in deactivated (EDTA) cerebral arterioles during a second SH. Captopril decreased the lower limit of CBF autoregulation (61+/-6 in SHR-6C and 51+/-2 in SHR-15C vs 52+/-6 in WKY-6 and 62+/-7 in WKY-15 and 83+/-14 mmHg in SHR-6 and 120+/-19 mmHg in SHR-15; P<0.05) and CSA (510+/-21 in SHR-6C and 585+/-25 in SHR-15C vs 529+/-12 in WKY-6 and 549+/-20 in WKY-15 and 644+/-38 mmHg in SHR-6 and 704+/-38 mmHg in SHR-15; P<0.05). Captopril increased cerebral arteriolar external diameter of SHR (105+/-5 in SHR-6C and 94+/-4 in SHR-15C vs 125+/-8 in WKY-6 and 108+/-3 in WKY-15 and 83+/-2 mmHg in SHR-6 and 80+/-2 mmHg in SHR-15 for a pial arteriolar pressure step of 35-39 mmHg; P<0.05). Captopril attenuated increases in cerebral arteriolar distensibility in young SHR. Thus, ACEIs attenuate eutrophic and hypertrophic inward remodeling of cerebral arterioles in young and old SHR, thus decreasing the lower limit of CBF autoregulation.

Aging↗

PubMed2005

Comparative effects of the angiotensin II receptor blocker, telmisartan, and the angiotensin-converting enzyme inhibitor, ramipril, on cerebrovascular structure in spontaneously hypertensive rats.

OBJECTIVE: Antihypertensive treatment with angiotensin-converting enzyme inhibitors (ACEIs) reverses cerebral arteriolar remodeling, thus restoring dilatation and hence the lower limit of cerebral blood flow (CBF) autoregulation (LLCBF). The objective of this study was to determine whether angiotensin II receptor AT1 blockers (ARBs) produce the same effect. DESIGN: We examined the effects of treatment with an ARB [telmisartan (TEL), 1.93 +/- 0.04 mg/kg per day] or an ACEI [ramipril (RAM), 1.00 +/- 0.02 mg/kg per day] on the cerebral circulation in spontaneously hypertensive rats (SHR). METHODS: Arteriolar pressure and diameter (cranial window) and CBF (laser Doppler) were measured during stepwise hypotensive hemorrhage, before and after deactivation (ethylenediamine tetraacetic acid), in untreated Wistar-Kyoto (WKY) rats and SHR untreated or treated for 3 months with TEL or RAM in the drinking water. RESULTS: Treatment normalized arteriolar internal diameter (SHR, 38 +/- 3 microm; TEL, 52 +/- 2 microm; RAM, 50 +/- 2 microm; WKY, 58 +/- 4 microm), essentially by reversing eutrophic inward remodeling, and the LLCBF (SHR, 80 +/- 11 mmHg; TEL, 60 +/- 4 mmHg; RAM, 71 +/- 6 mmHg; WKY, 57 +/- 5 mmHg). CONCLUSION: The fact that the ARB (TEL) is as effective as an ACEI (RAM) in reversing cerebral arteriolar remodeling suggests that the cerebrovascular AT1 receptor is an underlying mechanism that promotes hypertensive eutrophic inward remodeling.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2005

Oxidative stress activates MMP-2 in cultured human coronary smooth muscle cells.

Oxidative stress is a cardinal feature of the inflammatory process and is involved in various pathologies including atherosclerosis. One of the important mechanisms in which oxidative stress may play a role is activation of matrix metalloproteinases such as MMP-2, which are involved in plaque destabilization. We investigated the mechanisms by which oxidative stress induces MMP-2 activation in cultured human coronary artery smooth muscle cells. Using zymography and Western blot analysis, we showed that oxidized low-density lipoproteins activate MMP-2 through up-regulation of the expression and activation of a membrane-type 1 matrix metalloproteinase (MT1-MMP). A second mechanism of MMP-2 activation involves oxidative radicals generated by the xanthine/xanthine oxidase complex (X/Xo). Research on these two mechanisms of MMP activation could lead to the elaboration of new vascular therapies for the treatment of atheroma based on interruption of a specific oxidative stress pathway.

Cells, Cultured↗

PubMed2004

Incubation of rat aortic rings produces a specific reduction in agonist-evoked contraction: effect of age of donor.

We studied the effect of age on the response of aortic rings to injury produced by three days' incubation, and the mechanism of this response. Five-mm rings of the thoracic aorta isolated from Wistar rats were incubated or not in culture medium. Isometric contraction evoked by agonists (norepinephrine or serotonin) or high [K(+)](e) was determined in the presence and absence of endothelium. Experiments were repeated in the presence of propranolol (0.3 microM), polymixin B (36 microM), pyrrolidine dithiocarbamate (50 microM) or glutathione (3 mM). Inductible NO-synthase and cyclo-oxygenase-2 mRNA were determined by real-time PCR, and glutathione-related enzymes and catalase activity by spectrophotometry. Incubation reduced the isometric contraction evoked by agonists but not by high [K(+)](e). The reduction in agonist-evoked contraction was greater in rings from adult (norepinephrine Emax-80%) than in young (-40%) rats. The removal of the endothelium had no effect. The reduction in norepinephrine-evoked contraction was not due to endotoxin contamination, beta-adrenoceptor-mediated dilation or any change in ring structure (no fibrosis or edema). Inductible NO-synthase (but not cyclo-oxygenase-2) mRNA increased on incubation. N(G)-nitro-L-arginine methyl ester partially restored contractility in rings from adult animals, further addition of an anti-oxidant restored norepinephrine-evoked contraction. Catalase fell with age and glutathione reductase increased upon incubation in rings from young donors only. In conclusion, incubation of the aorta produces a specific reduction in agonist-evoked contraction that involves induction of smooth muscle cell oxidative stress and iNOS. The reaction is greater in rings from older animals.

Age Factors↗

PubMed2004

Differential response of human ovarian cancer cells to induction of apoptosis by vitamin E Succinate and vitamin E analogue, alpha-TEA.

A vitamin E derivative, vitamin E succinate (VES; RRR-alpha-tocopheryl succinate), and a vitamin E analogue, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxy acetic acid (alpha-TEA), induce human breast, prostate, colon, lung, cervical, and endometrial tumor cells in culture to undergo apoptosis but not normal human mammary epithelial cells, immortalized, nontumorigenic breast cells, or normal human prostate epithelial cells. Human ovarian and cervical cancer cell lines are exceptions, with alpha-TEA exhibiting greater proapoptotic effects. Although both VES and alpha-TEA can induce A2780 and subline A2780/cp70 ovarian cancer cells to undergo DNA synthesis arrest within 24 h of treatment, only alpha-TEA is an effective inducer of apoptosis. VES or alpha-TEA treatment of cp70 cells with 5, 10, or 20 microg/ml for 3 days induced 5, 6, and 19% versus 9, 36, and 71% apoptosis, respectively. Colony formation data provide additional evidence that cp70 cells are more sensitive to growth inhibition by alpha-TEA than VES. Differences in stability of the ester-linked succinate moiety of VES versus the ether-linked acetic acid moiety of alpha-TEA were demonstrated by high-performance liquid chromatography analyses that showed alpha-TEA to remain intact, whereas VES was hydrolyzed to the free phenol, RRR-alpha-tocopherol. Pretreatment of cp70 cells with bis-(p-nitrophenyl) phosphate, an esterase inhibitor, before VES treatment, resulted in increased levels of intact VES and apoptosis. Taken together, these data show alpha-TEA to be a potent and stable proapoptotic agent for human ovarian tumor cells and suggest that endogenous ovarian esterases can hydrolyze the succinate moiety of VES, yielding RRR-alpha-tocopherol, an ineffective apoptotic-inducing agent.

Apoptosis↗

PubMed2004

Molecular determinants of heritable vitamin E deficiency.

Tocopherol transfer protein (TTP) is a key regulator of vitamin E homeostasis. TTP is presumed to function by transporting the hydrophobic vitamin between cellular compartments, thus facilitating its secretion to the extracellular space. Indeed, recombinant TTP demonstrates marked ability to facilitate tocopherol transfer between lipid bilayers. We report the biochemical characterization of six missense mutations TTP(1) that are found in human AVED patients. We expressed the H101Q, A120T, R192H, R59W, E141K, and R221W TTP mutants in Escherichia coli, and purified the proteins to homogeneity. We then characterized TTP and its mutant counterparts with respect to their affinity for RRR-alpha-tocopherol and to their ability to catalyze tocopherol transfer between membranes. We observe the R59W, E141K, and R221W mutations, associated with the severe, early-onset version of AVED, are impaired in tocopherol binding and transfer activity. Surprisingly, despite the profound clinical effect of the R59W, E141K, and R221W mutations in vivo, their impact on TTP activity in vitro is quite benign (2-3-fold reduction in transfer kinetics). Furthermore, mutations associated with milder forms of the AVED disease, while causing pronounced perturbations in tocopherol homeostasis in vivo, are remarkably similar to the wild-type protein in the tocopherol transfer assays in vitro. Our data indicate that tocopherol transfer activity in vitro does not properly recapitulate the physiological functions of TTP. These findings suggest the possibility that the AVED syndrome may not arise from an inability of TTP to bind or to transfer alpha tocopherol, but rather from defects in other activities of the protein.

Amino Acid Substitution↗

PubMed2004

Impact of treatment with melatonin on cerebral circulation in old rats.

Melatonin deprival in young rats induces alterations in cerebral arteriolar wall similar to those observed during aging: atrophy and a decrease in distensibility. In this study, we examined the effects of melatonin treatment on cerebral arteriolar structure and distensibility and on the lower limit of cerebral blood flow autoregulation (LLCBF) in old rats. We measured cerebral blood flow (arbitrary unit, laser Doppler, open skull preparation) prior to and during stepwise hypotension (SH) in adult (12/13 months) and old (24/25 months) IcoWI and WAG/Rij male rats. Old rats were untreated or treated for 3 months with melatonin (0.39 (IcoWi) and 0.44 (Wag/Rij) mg kg-1 day-1, drinking water). Stress-strain relationships were determined using cross-sectional area (CSA, microm2, histometry) and values of arteriolar internal diameter (microm) obtained during a second SH following arteriolar deactivation (EDTA, 67 mmol l(-1)). Aging induced (a) atrophy of the arteriolar wall in IcoWI (616+/-20 vs 500+/-27 microm2, P<0.05) but not in WAG/Rij rats (328+/-25 vs 341+/-20 microm2), (b) a decrease in arteriolar wall distensibility and (c) an increase in the LLCBF in both strains (67+/-10 mmHg in 12-month-old vs 95+/-6 mmHg in 24-month-old IcoWi, P<0.05 and 53+/-2 mmHg in 13-month-old vs 67+/-6 mmHg in 25-month-old WAG/Rij). Melatonin treatment induced in IcoWI and WAG/Rij rats (a) hypertrophy of the arteriolar wall (643+/-34 and 435+/-25 microm2, respectively), (b) an increase in arteriolar wall distensibility and (c) a decrease in the LLCBF (64+/-6 and 45+/-4 mmHg, respectively). Melatonin treatment of old rats induced hypertrophy of the arteriolar wall, prevented the age-linked decrease in cerebral arteriolar distensibility and decreased the LLCBF. British Journal of Pharmacology (2004) 141, 399-406. doi:10.1038/sj.bjp.0705629

Aging↗