The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis.
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In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett. 418, 297-300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions. (+info)
Relationship between gastro-intestinal complaints and endotoxaemia, cytokine release and the acute-phase reaction during and after a long-distance triathlon in highly trained men.
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The aim of the present study was to establish whether gastro-intestinal (GI) complaints observed during and after ultra-endurance exercise are related to gut ischaemia-associated leakage of endotoxins [lipopolysaccharide (LPS)] into the circulation and associated cytokine production. Therefore we collected blood samples from 29 athletes before, immediately after, and 1, 2 and 16 h after a long-distance triathlon for measurement of LPS, tumour necrosis factor-alpha and interleukin-6 (IL-6). As the cytokine response would trigger an acute-phase response, characteristic variables of these responses were also measured, along with creatine kinase (CK) to obtain an indicator of muscle damage. There was a high incidence (93% of all participants) of GI symptoms; 45% reported severe complaints and 7% of the participants abandoned the race because of severe GI distress. Mild endotoxaemia (5-15 pg/ml) was evident in 68% of the athletes immediately after the race, as also indicated by a reduction in IgG anti-LPS levels. In addition, we observed production of IL-6 (27-fold increase immediately after the race), leading to an acute-phase response (20-fold increase in C-reactive protein and 12% decrease in pre-albumin 16 h after the race). The extent of endotoxaemia was not correlated with the GI complaints or the IL-6 response, but did show a correlation with the elevation in C-reactive protein (r(s) 0.389; P=0.037). Creatine kinase levels were increased significantly immediately post-race, and increased further in the follow-up period. Creatine kinase levels did not correlate with those of either IL-6 or C-reactive protein. It is therefore concluded that LPS does enter the circulation after ultra-endurance exercise and may, together with muscle damage, be responsible for the increased cytokine response and hence GI complaints in these athletes. (+info)
Transthyretin in high density lipoproteins: association with apolipoprotein A-I.
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Previous studies have revealed the presence of transthyretin (TTR) on lipoproteins. To further address this issue, we fractionated plasma lipoproteins from 9 normal individuals, 10 familial amyloidotic polyneuropathy (FAP) patients, and 19 hyperlipidemic subjects using gel filtration. In the majority of the subjects, as well as in 9 of the 10 FAP patients and 14 of the 19 patients with hyperlipidemia, TTR was detected by ELISA in the high density lipoprotein (HDL) fraction. The presence of TTR in HDL was confirmed by direct sequencing and by immunoblotting; using non-reducing conditions, TTR was found by immunoblotting in a high molecular weight complex, which reacted also for apolipoprotein A-I (apoA-I). The amount of TTR present in HDL (HDL-TTR), as quantified by ELISA corresponded to 1;-2% of total plasma TTR. However, no detectable TTR levels were found in HDL fraction from 6 of the hyperlipidemic subjects. No correlation was found between the lack of TTR in HDL and plasma levels of total, LDL-, or HDL-associated cholesterol as well as levels of apoA-I and total plasma TTR. Ligand binding experiments showed that radiolabeled TTR binds to the HDL fraction of individuals with HDL-TTR but not to the corresponding fractions of individuals devoid of HDL-TTR, suggesting that HDL composition may interfere with TTR binding. The component(s) to which TTR binds in the HDL fraction were investigated. Polyclonal antibody against apoA-I was able to block the interaction of TTR with HDL, suggesting that the interaction of TTR with the HDL particle occurs via apoA-I. This hypothesis was further demonstrated by showing the formation of a complex of TTR with HDL and apoA-I by crosslinking experiments. Furthermore, anti-apoA-I immunoblot under native conditions suggested the existence of differences in HDL particle properties and/or stability between individuals with and without HDL-TTR. (+info)
Effect of vitamin A status at the end of term pregnancy on the saturation of retinol binding protein with retinol.
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BACKGROUND: Vitamin A (retinol), which is required for normal fetal development and successful gestation, circulates in the blood bound to a specific protein, the retinol binding protein (RBP). Little is known about the transport and metabolism of this complex protein or about retinol status during normal human pregnancy. OBJECTIVE: The aim of this study was to assess retinol status and transport modalities of retinol in well-nourished women with normal pregnancies, a population poorly investigated compared with pathologic and malnourished pregnant women. DESIGN: The maternal blood and cord blood concentrations of retinol, vitamin E, beta-carotene, RBP, and transthyretin of pregnant French women at term (n = 27) were measured and compared with values from a nonpregnant control group (n = 27). In addition, holo-RBP (retinol bound), apo-RBP (retinol free), and total protein were assessed in both groups to enable the hemodilution occurring during pregnancy to be taken into consideration and to evaluate the extent of saturation of RBP with retinol. RESULTS: Healthy pregnant women at term had normal serum circulatory amounts of retinol, vitamin E, binding proteins, and beta-carotene. However, they had less binding of retinol to RBP (holo-RBP: 49.9% in pregnant women, 54.0% in cord blood, and 77.5% in the control group). CONCLUSION: The results of this study suggest that retinol homeostasis and transport are modified during normal human pregnancy. (+info)
Nicotine enhances the biosynthesis and secretion of transthyretin from the choroid plexus in rats: implications for beta-amyloid formation.
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Epidemiological studies indicated that cigarette smoking protects against the development of several neurodegenerative disorders, including Alzheimer's disease (AD). However, the molecular mechanism(s) underlying this is poorly understood. To gain insight into these protective effects, we used differential display PCR (DD-PCR) to amplify RNA from various brain regions of rats self-administering (SA) nicotine compared with yoked-saline controls. We found that the transthyretin (TTR) gene, whose product has been shown to bind to amyloid beta (Abeta) protein and prevent Abeta aggregation, was more abundantly expressed ( approximately 1.5- to 2.0-fold) in the brainstem and hippocampus (areas containing choroid plexus) of nicotine SA rats. Subsequently, quantitative reverse transcription-PCR analysis confirmed these DD-PCR findings and demonstrated that nicotine increased TTR mRNA levels in these regions in a time- and dose-dependent manner. Significantly higher TTR protein concentrations were also detected in the ventricular CSF of nicotine-treated rats. In contrast, no differences either in plasma TTR or in CSF and plasma retinol-binding protein were detected. Immunohistochemical analysis showed that immunoreactive TTR was 41.5% lower in the choroid plexus of nicotine-treated rats compared with the saline controls. On the basis of these data, we speculate that the protective effects of nicotine on the development of AD may be attributable, in part, to the increased biosynthesis and secretion of TTR from the choroid plexus. These findings also point toward new approaches that may take advantage of the potentially novel therapeutic effects of nicotinic agonists in patients with AD. (+info)
Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: a clinical application of mass spectrometry.
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In our continuing efforts to develop mass spectrometry-based methods for transthyretin (TTR) variant detection and characterization, we have sought to use matrix-assisted laser desorption/ionization (MALDI) bioreactive probes incorporating immobilized trypsin for screening purposes. These devices show good diagnostic potential as a clinical screening tool to detect amino acid substitutions in TTR. MALDI probes allow the on-probe generation of tryptic digests. The subsequent mass analysis of the on-probe digest yields the peptide map. The inherent advantages of this method include considerably reduced digestion times (minutes vs. hours), absence of autolysis products, minimized sample handling, and hence minimal sample loss. A further advantage is that the opportunity for loss of hydrophobic peptides is reduced because no sample transfer occurs. The method can be applied as a preliminary screen for TTR variants where TTR is isolated from patient serum through immunoprecipitation. This method should also be applicable to other proteins and suitable for automation. (+info)
Iron and zinc supplementation improves indicators of vitamin A status of Mexican preschoolers.
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BACKGROUND: The coexistence of multiple micronutrient deficiencies is a widespread public health problem in many regions of the world. Interactions between zinc deficiency and vitamin A metabolism have been reported but no longitudinal studies have evaluated the effect of iron deficiency on vitamin A. OBJECTIVE: The objective of this study was to investigate the effect of supplementation with iron, zinc, or both on vitamin A and its metabolically related proteins retinol binding protein (RBP) and transthyretin. DESIGN: The study was a longitudinal, double-blind, placebo-controlled trial in which 219 rural Mexican children aged 18-36 mo were randomly assigned to receive 20 mg Zn/d, 20 mg Fe/d, 20 mg Zn/d plus 20 mg Fe/d, or placebo. RESULTS: Six months after supplementation, plasma retinol increased in all supplemented groups. Compared with placebo, zinc supplementation was associated with significantly higher plasma retinol and transthyretin but the increase in RBP was not significant. Iron supplementation significantly increased plasma retinol, RBP, and transthyretin. Supplementation with zinc plus iron significantly increased plasma retinol but not RBP or transthyretin. Children deficient in zinc, iron, or vitamin A (as indicated by nutrient plasma concentration) at the beginning of the study had a significantly greater increase in retinol than did children with adequate nutrient status. CONCLUSIONS: Supplementation with zinc, iron, or both improved indicators of vitamin A status. The results of this study agree with previous observations of a metabolic interaction between zinc and vitamin A and suggest an interaction between iron and vitamin A metabolism. (+info)
Vintage, nutritional status, and survival in hemodialysis patients.
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BACKGROUND: The link between dialysis "vintage" (length of time on dialysis in months to years) and survival has been difficult to define, largely because of selection effects. End-stage renal disease (ESRD) is thought to be a wasting illness, but there are no published reports describing the associations between vintage and body composition in hemodialysis patients. METHODS: We explored the relationships among vintage, nutritional status, and survival in a 3009 patient cohort of prevalent hemodialysis patients. Body weight, total body water, body cell mass, and phase angle by bioelectrical impedance analysis were the body composition parameters of interest. We examined vintage as an explanatory variable in multiple linear regression analyses (adjusted for age, gender, race, and diabetes) using body composition parameters and biochemical indicators of nutritional status as dependent variables. Proportional hazards regression was used to evaluate the association of vintage and survival with and without adjustment for case mix and laboratory variables. RESULTS: Dialysis vintage was 3.8 +/- 3.7 (median 2.6) years. Body composition parameters tended to be lower after dialysis year 2. Linear estimates per year of vintage beyond year 2 include -0.66 kg body wt (P < 0.0001), -0.17 kg total body water (P = 0.0003), -0.14 kg body cell mass (P < 0.0001), and -0.07 degrees phase angle (P < 0.0001). In unadjusted analyses, vintage was not associated with survival, either as a linear or higher order term. The adjustment for case mix yielded a vintage term associated with an increased relative risk (RR) of death (RR 1.04 (95% CI, 1.01 to 1.07 per year). A further adjustment for laboratory data yielded a RR of 1.06 (95% CI, 1.03 to 1.09 per year). CONCLUSION: Dialysis vintage is related to nutritional status in hemodialysis patients, with vintage of more than years associated with a significant decline in all measured nutritional parameters. Cross-sectional analyses probably underestimate these effects. A year accrued on dialysis is associated with a 6% increase in the risk of death, all else equal. Longitudinal assessments of nutritional status, including body composition, are required to better understand the natural history of wasting with ESRD and its implications for long-term survival. (+info)