The autoantigen Ro52 is an E3 ligase resident in the cytoplasm but enters the nucleus upon cellular exposure to nitric oxide.

Patients with the systemic autoimmune diseases Sjögrens's syndrome and systemic lupus erythematosus often have autoantibodies against the intracellular protein Ro52. Ro52 is an E3 ligase dependent on the ubiquitin conjugation enzymes UBE2D1 and UBE2E1. While Ro52 and UBE2D1 are cytoplasmic proteins, UBE2E1 is localized to the nucleus. Here, we investigate how domains of human Ro52 regulate its intracellular localization. By expressing fluorescently labeled Ro52 and Ro52 mutants in HeLa cells, an intact coiled-coil domain was found to be necessary for the cytoplasmic localization of Ro52. The amino acids 381-470 of the B30.2 region were essential for translocation into the nucleus. Furthermore, after exposure of HeLa cells to the inflammatory mediator nitric oxide (NO), Ro52 translocated to the nucleus. A nuclear localization of Ro52 in inflamed tissue expressing inducible NO synthetase (iNOS) from cutaneous lupus patients was observed by immunohistochemistry and verified in NO-treated cultures of patient-derived primary keratinocytes. Our results show that the localization of Ro52 is regulated by endogenous sequences, and that nuclear translocation is induced by an inflammatory mediator. This suggests that Ro52 has both cytoplasmic and nuclear substrates, and that Ro52 mediates ubiquitination through UBE2D1 in the cytoplasm and through UBE2E1 in the nucleus.

Drinking water supplementation with ascorbate is not protective against UVR-B-induced cataract in the guinea pig.

PURPOSE: To study if ascorbate supplementation decreases ultraviolet radiation (UVR)-induced cataract development in the guinea pig. METHODS: Sixty 6-9-week-old pigmented guinea pigs received drinking water supplemented with or without 5.5 mm l-ascorbate for 4 weeks. After supplementation, 40 animals were exposed unilaterally in vivo under anaesthesia to 80 kJ/m(2) UVR-B. One day later, the animals were killed and lenses were extracted. Degree of cataract was quantified by measurement of intensity of forward lens light scattering. Lens ascorbate concentration was determined with high-performance liquid chromatography (HPLC) with UVR detection at 254 nm. Twenty animals were used as non-exposed control. RESULTS: Supplementation increased lens ascorbate concentration significantly. In UVR-exposed animals, mean 95% confidence intervals (CIs) for animal-averaged lens ascorbate concentration (micromol/g wet weight lens) were 0.54 +/- 0.07 (no ascorbate) and 0.83 +/- 0.05 (5.5 mm ascorbate). In non-exposed control animals, mean 95% CIs for animal-averaged lens ascorbate concentration (micromol/g wet weight lens) were 0.72 +/- 0.12 (0 mm ascorbate) and 0.90 +/- 0.15 (5.5 mm ascorbate). All non-exposed lenses were devoid of cataract. Superficial anterior cataract developed in all UVR-exposed lenses. The lens light scattering was 39.2 +/- 14.1 milli transformed equivalent diazepam concentration (m(tEDC)) without and 35.9 +/- 14.0 m(tEDC) with ascorbate supplementation. CONCLUSION: Superficial anterior cataract develops in lenses exposed to UVR-B. Ascorbate supplementation is non-toxic to both UVR-B-exposed lenses and non-exposed control lenses. Ascorbate supplementation does not reduce in vivo lens forward light scattering secondary to UVR-B exposure in the guinea pig.

Interferon-alpha induces up-regulation and nuclear translocation of the Ro52 autoantigen as detected by a panel of novel Ro52-specific monoclonal antibodies.

Interferon-alpha (IFN-alpha) has been implicated in the pathogenesis of Sjögren's syndrome and systemic lupus erythematosus. Ro52, which was recently identified as an E3 ligase with anti-proliferative and pro-apoptotic properties, is a major autoantigen targeted in both these conditions. Microarray analyses have indicated up-regulation of Ro52 by IFN-alpha, and the objective of the present study was to address the potential link between IFN-alpha and Ro52. To investigate the influence of IFN-alpha on Ro52 protein levels and cellular localization, we generated a panel of monoclonal antibodies to different domains of Ro52. These novel monoclonal antibodies were characterized by immunoprecipitation, Western blot, and enzyme-linked immunosorbent assay using cell lysates, recombinant Ro52 protein, and synthetic peptides. Ro52 was up-regulated in HeLa cells and human B cells at the messenger RNA and protein levels in response to IFN-alpha stimulation as detected by reverse transcriptase polymerase chain reaction and Western blot. After up-regulation, Ro52 translocated from the cytoplasm to the nucleus. The nuclear translocation of Ro52 was observed after staining with generated monoclonal antibodies specific for both the RING, coiled-coil, and B30.2 domains of Ro52 and the nuclear translocation of Ro52 preceded IFN-alpha-induced apoptotic cell death detected by caspase-3 and TUNEL staining in the treated cultures. In conclusion, our data show that IFN-alpha first induces up-regulation of Ro52 protein and then prompts translocation of the up-regulated Ro52 protein in to the nucleus. The translocation precedes apoptosis of the IFN-alpha exposed cells, suggesting a role for Ro52 in mediating the anti-proliferative or pro-apoptotic effects of the autoimmune-related cytokine IFN-alpha.

Ultraviolet radiation-B-induced cataract in albino rats: maximum tolerable dose and ascorbate consumption.

PURPOSE: To investigate the maximum tolerable dose (MTD) for cataract induced by ultraviolet radiation-B (UVB) in 7-week-old albino rats and to study the effect of UVB eye exposure on lens ascorbate content. METHODS: Fifty 7-week-old albino Sprague Dawley rats were unilaterally exposed in vivo to 300-nm UVB under anaesthesia, receiving 0, 0.25, 3.5, 4.3 and 4.9 kJ/m(2). The MTD was estimated based on lens forward light scattering measurements. Lens ascorbate content was determined in the processed lens using high performance liquid chromatography with UVR detection. RESULTS: Animals exposed to UVB doses >or=3.5 kJ/m(2) developed cortical cataracts. The MTD for avoidance of UVB-induced cataract was estimated to 3.01 kJ/m(2). UVB exposure decreased lens ascorbate concentration in the exposed lens in line with UVB dose, H(e), according to the models: C = C(NonCo) + C(Co)e(-kH(e) ) for exposed lenses; C = C(NonCo) + C(Co) for non-exposed lenses, and C(d) = C(Co)(e(-kH(e) ) - 1). Parameters for consumable and non-consumable ascorbate were estimated to C(NonCo) = 0.04 and C(Co) = 0.11 micromol/g wet weight of lens. For lens ascorbate difference, tau = 1/k = 0.86 kJ/m(2). A total of 63% of UVB consumable ascorbate has been consumed after only tau = 0.86 kJ/m(2), while MTD(2.3 : 16) = 3.01 kJ/m(2), indicating that ascorbate decrease is in the order of 3.5 times more sensitive to detecting UVR damage in the lens than forward light scattering. CONCLUSIONS: The MTD for avoidance of UVB-induced cataract in the 7-week-old albino Sprague Dawley rat was estimated to be 3.01 kJ/m(2). In vivo UVB exposure of the rat eye decreases lens ascorbate content following an exponential decline, and suprathreshold doses cause greater effect than subthreshold doses.

The ketogenic diet influences the levels of excitatory and inhibitory amino acids in the CSF in children with refractory epilepsy.

The ketogenic diet (KD) is an established treatment for medically refractory pediatric epilepsy. Its anticonvulsant mechanism is still unclear. We examined the influence of the KD on the CSF levels of excitatory and inhibitory amino acids in 26 children (mean age 6.1 years) with refractory epilepsy. Seventeen amino acids were determined before and at a mean of 4 months after the start of the KD. Seizures were quantified. Highly significant changes were found in eight amino acids: increases in GABA, taurine, serine, and glycine and decreases in asparagine, alanine, tyrosine and phenylalanine. However, aspartate, glutamate, arginine, threonine, citrulline, leucine, isoleucine and valine/methionine remained unchanged. A significant correlation with seizure response was found for threonine (P=0.016). The GABA levels were higher in responders (>50% seizure reduction) than in nonresponders during the diet (P=0.041). In the very good responders (>90% seizure reduction), the GABA levels were significantly higher at baseline as well as during the diet. Age differences were found with significantly larger decreases in glutamate and increases in GABA in connection with the diet in younger children. Our results indicate that the KD significantly alters the levels of several CSF amino acids that may be involved in its mechanism of action and the increase in GABA is of particular interest.

Ascorbate in the rat lens: dependence on dietary intake.

PURPOSE: To establish a method for sample preparation to measure ascorbate in whole lenses and to investigate whether lens ascorbate concentration is dependent on dietary ascorbate intake. METHODS: Four groups of 3 young Sprague-Dawley rats each were fed chow containing L-ascorbate, either 0.0, 5.7, 57.0 or 114.0 mmol/kg for a duration of 4 weeks. Thereafter, each rat was sacrificed. The lens was extracted, photographed, and lens wet weight was measured. The lens was homogenized in 1.0 ml of 0.25% metaphosphoric acid, the homogenate was centrifuged and the supernatant ultrafiltered. The filtrate was injected into an ion exchange, reversed-phase Polypore H HPLC column equipped with a 254-nm ultraviolet detector. Samples were calibrated against an L-ascorbate standard. Polynomial regression analysis was performed on the data. RESULTS: All lenses were devoid of cataract. A 95% confidence interval for baseline content of ascorbate without any dietary intake was estimated to be 0.16+/-0.01 micromol/g wet weight of lens. The lens ascorbate concentration increased linearly with dietary ascorbate intake with an increased rate, estimated as a 95% confidence interval of 0.33+/-0.18 (micromol ascorbate) (g lens)-1)(mol ascorbate)-1 (kg chow) with r2=0.62. CONCLUSION: Lens ascorbate concentration linearly increases with dietary ascorbate intake without cataract development in the rat. The currently presented method for sample preparation to measure the whole-lens content of ascorbate is applicable.

Ascorbate in the guinea pig lens: dependence on drinking water supplementation.

PURPOSE: To investigate whether lens ascorbate concentration can be elevated with drinking water supplementation. METHODS: Pigmented guinea pigs received drinking water supplemented with L-ascorbate, concentration 0.00, 2.84, 5.68 or 8.52 mm for a duration of 4 weeks. In addition, the chow fed to all animals contained 125 mmol L-ascorbate per kg of chow. At the end of the supplementation period, the guinea pigs were killed. Each lens was extracted. The lens was processed and ascorbate concentration was measured using high performance liquid chromatography (HPLC) with 254 nm ultraviolet radiation detection. The data were analysed with regression. RESULTS: At the end of the test period, all lenses were devoid of cataract as observed by slit-lamp examination. All lenses contained a detectable concentration of ascorbate. Estimated 95% confidence intervals for mean animal-averaged lens ascorbate concentrations (micromol/g wet weight of whole lens) per group were 0.51 +/- 0.04 (0.00 mm; n = 6), 0.70 +/- 0.18 (2.84 mm; n = 6), 0.71 +/- 0.11 (5.68 mm; n = 5), and 0.71 +/- 0.06 (8.52 mm; n = 6). Animal-averaged lens ascorbate concentration [Asc(lens)] (micromol/g wet weight lens) increased with ascorbate supplementation in drinking water [Asc(water)] (M), in agreement with the model: [Asc(lens)] = A - Be(-kAsc(water)]. CONCLUSION: Lens ascorbate concentration increases with drinking water supplementation in the guinea pig without cataract development. The currently presented method for measurement of whole lens ascorbate content is suitable.

The effects of PBN (phenyl-butyl-nitrone) on GLT-1 levels and on the extracellular levels of amino acids and energy metabolites in a model of iron-induced posttraumatic epilepsy.

This study investigates astrocytic glutamate uptake in the iron-induced animal model of posttraumatic epilepsy. Since formation of free radicals may be involved in epileptogenesis after brain trauma and hemorrhage the effects of the nitrone radical scavenger alpha-phenyl-tert-N-butyl nitrone (PBN) were also studied. Animals received an intracortical iron injection, or were sham-operated. They were given PBN intraperitoneally or saline as control. Twenty-four hours after lesion, brain tissue was collected and the level of glial glutamate transporter (GLT-1) was analyzed using immunoblotting. The extracellular concentrations of amino acids and energy metabolites were measured using microdialysis. The results showed significantly decreased levels of GLT-1 (70 kDa), higher basal levels of glutamate, and lower levels of glutamine as well as low arginine/citrulline ratios at the lesion compared to controls. PBN significantly attenuated the decrease of 70 kDa GLT-1 in the lesioned animals and attenuated the alterations in amino acid levels but not to a significant level. PBN also increased the arginine/citrulline ratios indicating reduced nitric oxide synthase activity. Our results suggest that astrocytic uptake of glutamate is oxidatively impaired in iron-induced epileptogenesis and that the administration of a radical scavenger can attenuate this process.