2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins

Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.

The sodium-dependent ascorbic acid transporter family SLC23

Transporters for vitamin C and its oxidized form dehydroascorbic acid (DHA) are crucial to maintain physiological concentrations of this important vitamin that is used in a variety of biochemical processes. The human SLC23 family consists of the Na(+)-dependent vitamin C transporters SVCT1 (encoded by the SLC23A1 gene) and SVCT2 (SLC23A2) as well as an orphan transporter SVCT3 (SLC23A3). Phylogenetically, the SLC23 family belongs to the nucleobase-ascorbate transporter (NAT) family, although no nucleobase transport has yet been demonstrated for the human members of this family. The SVCT1 and SVCT2 transporters are rather specific for ascorbic acid, which is an important antioxidant and plays a crucial role in a many metal-containing enzymes. SVCT1 is expressed predominantly in epithelial tissues such as intestine where it contributes to the supply and maintenance of whole-body ascorbic acid levels. In contrast to various other mammals, humans are not capable of synthesizing ascorbic acid from glucose and therefore the uptake of ascorbic acid from the diet via SVCT1 is essential for maintaining appropriate concentrations of vitamin C in the human body. The expression of SVCT2 is relatively widespread, where it serves to either deliver ascorbic acid to tissues with high demand of the vitamin for enzymatic reactions or to protect metabolically highly active cells or specialized tissues from oxidative stress. The murine Slc23a3 gene encoding the orphan transporter SVCT3 was originally cloned from mouse yolk sac, and subsequent studies showed that it is expressed in the kidney. However, the function of SVCT3 has not been reported and it remains speculative as to whether SVCT3 is a nucleobase transporter.

Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.

Expression, purification and low-resolution structure of human vitamin C transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.

Use of vitamin C in delayed tooth replantation

O objetivo deste trabalho foi avaliar microscopicamente, em reimplantes tardios de dentes de rato, os efeitos do tratamento da superfície radicular com diferentes soluções. Foram utilizados 30 ratos Rattus norvegicus albinos da linhagem Wistar que tiveram seus incisivos centrais extraídos e deixados sobre a bancada por 6 h. As polpas foram extirpadas e os canais irrigados com solução de hipoclorito de sódio a 1%. Após o preparo endodôntico, a superfície radicular de cada dente foi tratada com solução de hipoclorito de sódio a 1% por 10 min (trocada a cada 5 min) seguida de soro fisiológico por 10 min, e os dentes foram divididos em três grupos com 10 espécimes em cada um. Nos Grupos I, II e III, respectivamente, a superfície radicular foi tratada com fluoreto de sódio fosfato acidulado a 2%, vitamina C e vitamina C efervescente (2 g, Redoxon®). Após obturação com pasta de hidróxido de cálcio os dentes foram reimplantados e os animais foram sacrificados aos 10 e 60 dias. O Grupo I apresentou maiores áreas de reabsorção por substituição e anquilose. Comparando as formas de vitamina C utilizadas, a efervescente (Grupo III) foi a que apresentou resultados mais favoráveis com mais áreas de anquilose e reabsorção por substituição que áreas de reabsorção inflamatória.

Functional and physiological role of vitamin C transporters

Vitamin C (ascorbic acid) is required for the synthesis of collagen, carnitine, catecholamine and the neurotransmitter norepinephrine. Vitamin C also plays an important role in protection against oxidative stress. Transporters for vitamin C and its oxidized form dehydroascorbate (DHA) are crucial to keep vitamin concentrations optimal in the body. The human SLC23 family consists of the Na(+)-dependent vitamin C transporters SVCT1 (SLC23A1) and SVCT2 (SLC23A2) and the orphan transporter SVCT3 (SLC23A3). Phylogenetically, the SLC23 family belongs to the nucleobase-ascorbate transporter family although no specificity for nucleobases has yet been demonstrated for the human members of this family. In fact, the SVCT1 and SVCT2 transporters are rather specific for ascorbic acid. SVCT1 is expressed in epithelial tissues such as intestine, where it contributes to the maintenance of whole-body ascorbic acid levels, whereas the expression of SVCT2 is relatively widespread either to protect metabolically active cells and specialized tissues from oxidative stress or to deliver ascorbic acid to tissues that are in high demand of the vitamin for enzymatic reactions. DHA, the oxidized form of ascorbic acid is taken up and distributed in the body by facilitated transport via members of the SLC2/GLUT family (GLUT1, GLUT3, and GLUT4). Although, the main focus of this review is on the SLC23 family of ascorbic acid transporters, transporters of DHA and nucleobases are also briefly discussed for completeness.

In vivo vitamin C supplementation increases phosphoinositol transfer protein expression in peripheral blood mononuclear cells from healthy individuals

Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidylinositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression, in patients supplemented with vitamin C (0-2 g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2 g ascorbate/d after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of K+ channel proteins in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of action in vivo.

Abnormal changes in voltage-gated sodium channels NaV1.1, NaV1.2, NaV1.3, NaV1.6 and in Calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats

Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

The effect of 2 weeks vitamin C supplementation on immunoendocrine responses to 2.5 h cycling exercise in man

Davison G, Gleeson M, 2006. The effect of 2 weeks vitamin C supplementation on immunoendocrine responses to 2.5 h cycling exercise in man. European Journal of Applied Physiology 97(4): 454-461 RAE2008

Root surface treatment with vitamin C in tooth replantation: microscopic study in rats

AIM: Late tooth replantation is a worthy procedure, especially in growing patients, being the best option for tooth avulsion. This study evaluated the effects of root surface treatment with 2% acidulated phosphate sodium fluoride and effervescent vitamin C (Redoxon) in late replantation. METHODS: Twenty rat teeth (Rattus norvegicus, albinus, Wistar) were extracted and left on a table for 6 h. Then, the dental papilla and enamel organ were sectioned and the pulp was removed through the apex. After removal of the periodontal ligament with a blade, the animals were divided into 2 groups: Group I teeth were immersed in 2% acidulated phosphate sodium fluoride solution for 10 min and obturated with Ca(OH)(2) paste. Group II teeth were immersed in effervescent vitamin C solution (Redoxon 2 g) for 10 min and obturated as for Group I. After these procedures, teeth were replanted and animals were killed after 60 days. RESULTS: The study revealed a larger amount of replacement resorption in Group I and larger amount of ankylosis in Group II, with statistically significant difference and absence of inflammatory resorption. CONCLUSION: The substances used for root surface treatment were unable to prevent replacement resorption and ankylosis, which are expected when the periodontal ligament has been lost.

EFFECTS OF VITAMIN C SUPPLEMENTATION ON ACUTE PHASE CHAGAS DISEASE IN EXPERIMENTALLY INFECTED MICE WITH Trypanosoma cruzi QM1 STRAIN

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

Influence of Roux-en-Y Gastric Bypass Surgery on Vitamin C, Myeloperoxidase, and Oral Clinical Manifestations: A 2-Year Follow-Up Study

Background: Bariatric surgery influences the intake and absorption of nutrients. The serum concentrations of vitamin C, myeloperoxidase (MPO) and oral clinical manifestations were examined in patients two years after Roux-en-Y gastric bypass (RYGB). Methods: Clinical prospective-study with control-group (CG; n = 26), assessed only once, and the bariatric-group (BG; n = 26), assessed in the basal period and at 12 and 24 months after surgery. The mean ages in the CG and BG were 37.8 +/- 1.51 and 39.6 +/- 1.93 years, respectively, and their body mass indices were 22.07 +/- 0.29 and 45.62 +/- 1.46 kg/m2, respectively. Results: At 12 months after surgery, increased episodes of vomiting (P < .001) and dental hypersensitivity (P=.012) were observed, with a reduction in the saliva buffering capacity of 21.3 2.9% (P=.004). At 24 months after RYGB, we detected a significant reduction in serum vitamin C (32.9 +/- 5.3%, P < .001) and MPO values were higher than in the basal period (P = .032). With regard to oral hygiene habits, 92.3% of patients reported frequent tooth brushing and 96.1% used fluoride, which were similar across the two years. However, dental hypersensitivity (P = .048) was significantly increased than baseline. Conclusions: The results demonstrated that vitamin C deficiency and increased vomiting after RYGB for morbid obesity may contribute to increased periodontal disease. The fact it is impossible to determine which factors (diet, poor compliance with supplementation, vomiting, poor oral hygiene) contributed to the dental problems in these patients is a shortcoming of the report. (Nutr Clin Pract. 2012; 27: 114-121)

Vitamin C : effects of exercise and requirements with training

Ascorbic acid or vitamin C is involved in a number of biochemical pathways that are important to exercise metabolism and the health of exercising individuals. This review reports the results of studies investigating the requirement for vitamin C with exercise on the basis of dietary vitamin C intakes, the response to supplementation and alterations in plasma, serum, and leukocyte ascorbic acid concentration following both acute exercise and regular training. The possible physiological significance of changes in ascorbic acid with exercise is also addressed. Exercise generally causes a transient increase in circulating ascorbic acid in the hours following exercise, but a decline below pre-exercise levels occurs in the days after prolonged exercise. These changes could be associated with increased exercise-induced oxidative stress. On the basis of alterations in the concentration of ascorbic acid within the blood, it remains unclear if regular exercise increases the metabolism of vitamin C. However, the similar dietary intakes and responses to supplementation between athletes and nonathletes suggest that regular exercise does not increase the requirement for vitamin C in athletes. Two novel hypotheses are put forward to explain recent findings of attenuated levels of cortisol postexercise following supplementation with high doses of vitamin C.

Ternary metal complexes of anionic and neutral pyridoxine (vitamin B6) with 2,2'-bipyridine. Syntheses and x-ray structures of (pyridoxinato)bis(2,2'-bipyridyl)cobalt(III) perchlorate and chloro(2,2'-bipyridyl)(pyridoxine)copper(II) perchlorate hydrate

Ternary metal complexes involving vitamin B6 with formulas [CO",(PN-H)](anCdI [OC)'(bpy)(PN)Cl]C10(.bpHy 0 = 2,2'-bipyridine, PN = neutral pyridoxine, PN-H = anionic pyridoxine) have been prepared for the first time and characterized by means of magnetic and spectroscopic measurements. The crystal structures of the compounds have also been determined. [CO(PN-H)](CcryIsOta,l)lize s in the space group P2,/c with a = 18.900 (3) A, b = 8.764 (1) A, c = 20.041 (2) A,p = 116.05 (l)', and Z = 4 and [Cu(bpy)(PN)C1]C104-H20in the space group Pi with a = 12.136 (5) A, b = 13.283 (4) A,c = 7.195 (2) A, a = 96.91 (Z)', 0 = 91.25 (3)', y = 71.63 (3)', and Z = 2. The structures were solved by the heavy-atom method and refined by least-squares techniques to R values of 0.080 and 0.042 for 3401 and 2094 independent reflections, respectively. Both structures consist of monomeric units. The geometry around Co(II1) is octahedral and around Cu(I1) is distorted square pyramidal. In [CO(PN-H)]t(wCo IoxOy~ge)n~s ,fro m phenolic and 4-(hydroxymethyl) groups of PN-H and two nitrogens from each of two bpy's form the coordination sphere. In [Cu(bpy)(PN)C1]C104.H20o ne PN and one bpy, with the same donor sites, act as bidentate chelates in the basal plane, with a chloride ion occupying the apical position. In both structures PN and PN-H exist in the tautomeric form wherein pyridine N is protonated and phenolic 0 is deprotonated. However, a novel feature of the cobalt compound is that PN-H is anionic due to the deprotonation of the 4-(hydroxymethyl) group. The packing in both structures is governed by hydrogen bonds, and in the copper compound partial stacking of bpy's at a distance of -3.55 also adds to the stability of the system. Infrared, NMR, and ligand field spectroscopic results and magnetic measurements are interpreted in light of the structures.

Effects of Artemia enriched with unsaturated fatty acids and vitamin C on growth, survival and resistance Salmo trutta caspius larvae

This experiment was conducted to investigate the effect of using n-3 HUFA and Vitamin C enriched Artemia urmiana Nauplii Five difference treament were tested: for Caspian salmon (Salmo trutta caspius) larvae compare with artificial food in five treatment: (1) Artificial food, (2) Newly hatched Artemia (3) n-3 HUFA enriched Artemia (4) n-3 HUFA + 10% Ascorbyl Palmitate enriched Artemia (5) n-3 HUFA+20% Ascorbyl palmitate enriched Artemia during 15 days then all treatment were fed with artificial food during 20 days. In days of 15, larvae fed with newly hatched Artemia didn’t show significant difference of growth rate and survival compared to larvae fed with n-3 HUFA and Vitamn C enriched live food (p<0.05), However all treatment which fed live food have better growth rate and survival compred to larvae fed artificial food. Larvae fed with enriched Artemia with n-3 HUFA + 20% Ascorbyl palmitate has best result of temperature resistance at 26'C and 28'C. There is not significant difference between treatment (1) and (2), (3) and in this manner between (2), (3) and (4), (5) (P>0.05). In days of 35, larvae fed n-3 HUFA + 10% and 20% Ascorbyl pamlitate show better wet weight and dry weight compared to other treatment (P<0.05). Larvae fed n-3 HUFA Artemia showed significant difference compared to treatment (1) and (2), However there is not significant difference between treatment (1) and (2). Larvae fed artificial food show less and significant difference of survival compared to other treatment (P<0.05). Larvae fed artificial food show least of temperature resistance at 26'C and 28'C , However, there is not significant difference between all treatment (P<0.05).