A pilot study of neonatal GALT gene replacement using AAV9 dramatically lowers galactose metabolites in blood, liver, and brain and minimizes cataracts in GALT-null rat pups.

Classic galactosemia (CG) is a rare metabolic disorder that results from profound deficiency of galactose-1-P uridylyltransferase (GALT). Despite early detection by newborn screening and rapid and lifelong dietary restriction of galactose, which is the current standard of care, most patients grow to experience a broad constellation of long-term complications. The mechanisms underlying these complications remain unclear and likely differ by tissue. Here we conducted a pilot study testing the safety and efficacy of GALT gene replacement using our recently-described GALT-null rat model for CG. Specifically, we administered AAV9.CMV.HA-hGALT to seven GALT-null rat pups via tail vein injection on day 3 of life; eight GALT-null pups injected with PBS served as the negative control, and four GALT+ heterozygous pups injected with PBS served as the positive control. All pups were returned to their nursing mothers, weighed daily, and euthanized for tissue collection 2 weeks later. Among the AAV9-injected pups in this study, we achieved GALT levels in liver ranging from 64% to 595% wild-type, and in brain ranging from 3% to 42% wild-type. In liver, brain, and blood samples from these animals we also saw a striking drop in galactose, galactitol, and gal-1P. Finally, all treated GALT-null pups showed dramatic improvement in cataracts relative to their mock-treated counterparts. Combined, these results demonstrate that GALT restoration in both liver and brain of GALT-null rats by neonatal tail vein administration using AAV9 is not only attainable but effective.

Ten years of screening for congenital disorders of glycosylation in Argentina: case studies and pitfalls.

BACKGROUND: Congenital Disorders of Glycosylation (CDG) are genetic diseases caused by hypoglycosylation of glycoproteins and glycolipids. Most CDG are multisystem disorders with mild to severe involvement. METHODS: We studied 554 patients (2007-2017) with a clinical phenotype compatible with a CDG. Screening was performed by serum transferrin isoelectric focusing. The diagnosis was confirmed by genetic testing (Sanger or exome sequencing). RESULTS: A confirmed abnormal pattern was found in nine patients. Seven patients showed a type 1 pattern: four with PMM2-CDG, two with ALG2-CDG, and one with classical galactosemia. A type 2 pattern was found in two patients: one with a CDG-IIx and one with a transferrin protein variant. Abnormal transferrin pattern were observed in a patient with a myopathy due to a COL6A2 gene variant. CONCLUSIONS: CDG screening in Argentina from 2007 to 2017 revealed 4 PMM2-CDG patients, 2 ALG2-CDG patients with a novel homozygous gene variant and 1 CDG-IIx.

Antioxidant effects on the intracerebroventricular galactose damage in rats.

We investigated the effects of the intracerebroventricular infusion of galactose and the influence of pretreatment with antioxidants on oxidative stress parameters and acethylcholinesterase (AChE) activity in the brain of 60-day-old Wistar rats (6 per group). The animals were divided into naïve group (did not undergo surgery); procedure group (only underwent surgery); sham group (underwent surgery and received 5 µL saline) and galactose group (received 5 µL of galactose solution (5.0 mM) by intracerebroventricular injection), and were killed by decapitation after 1 h. Other groups were pretreated daily for 1 week with saline (sham and galactose groups) or antioxidants, α-tocopherol (40 mg/kg) plus ascorbic acid (100 mg/kg, i.p.) (antioxidants and galactose + antioxidants groups). Twelve hours after the last antioxidants injection, animals received an intracerebroventricular infusion of 5 µL of galactose solution (galactose and galactose + antioxidants groups) or saline (sham and antioxidants groups) and were sacrificed 1 h later. Galactose elevated thiobarbituric acid reactive substances (TBA-RS), protein carbonyl content and glutathione peroxidase (GSH-Px) activity and decreased total sulfhydryl content and catalase (CAT) activity in the cerebral cortex. In the hippocampus, galactose enhanced TBA-RS, decreased total sulfhydryl content and increased AChE activity, while in the cerebellum it decreased total sulfhydryl content and increased CAT and superoxide dismutase (SOD) activities. Pretreatment with antioxidants prevented the majority of these alterations, indicating the participation of free radicals in these effects. Thus, intracerebroventricular galactose infusion impairs redox homeostasis in the brain; the administration of antioxidants should be considered as an adjuvant therapy to specific diets in galactosemia.

HEPATOKIN1 is a biochemistry-based model of liver metabolism for applications in medicine and pharmacology.

The epidemic increase of non-alcoholic fatty liver diseases (NAFLD) requires a deeper understanding of the regulatory circuits controlling the response of liver metabolism to nutritional challenges, medical drugs, and genetic enzyme variants. As in vivo studies of human liver metabolism are encumbered with serious ethical and technical issues, we developed a comprehensive biochemistry-based kinetic model of the central liver metabolism including the regulation of enzyme activities by their reactants, allosteric effectors, and hormone-dependent phosphorylation. The utility of the model for basic research and applications in medicine and pharmacology is illustrated by simulating diurnal variations of the metabolic state of the liver at various perturbations caused by nutritional challenges (alcohol), drugs (valproate), and inherited enzyme disorders (galactosemia). Using proteomics data to scale maximal enzyme activities, the model is used to highlight differences in the metabolic functions of normal hepatocytes and malignant liver cells (adenoma and hepatocellular carcinoma).

Structural analysis of missense mutations in galactokinase 1 (GALK1) leading to galactosemia type-2.

Galactosemia type 2 is an autosomal recessive disorder characterized by the deficiency of galactokinase (GALK) enzyme due to missense mutations in GALK1 gene, which is associated with various manifestations such as hyper galactosemia and formation of cataracts. GALK enzyme catalyzes the adenosine triphosphate (ATP)-dependent phosphorylation of α-d-galactose to galactose-1-phosphate. We searched 4 different literature databases (Google Scholar, PubMed, PubMed Central, and Science Direct) and 3 gene-variant databases (Online Mendelian Inheritance in Man, Human Gene Mutation Database, and UniProt) to collect all the reported missense mutations associated with GALK deficiency. Our search strategy yielded 32 missense mutations. We used several computational tools (pathogenicity and stability, biophysical characterization, and physiochemical analyses) to prioritize the most significant mutations for further analyses. On the basis of the pathogenicity and stability predictions, 3 mutations (P28T, A198V, and L139P) were chosen to be tested further for physicochemical characterization, molecular docking, and simulation analyses. Molecular docking analysis revealed a decrease in interaction between the protein and ATP in all the 3 mutations, and molecular dynamic simulations of 50 ns showed a loss of stability and compactness in the mutant proteins. As the next step, comparative physicochemical changes of the native and the mutant proteins were carried out using essential dynamics. Overall, P28T and A198V were predicted to alter the structure and function of GALK protein when compared to the mutant L139P. This study demonstrates the power of computational analysis in variant classification and interpretation and provides a platform for developing targeted therapeutics.

Reversal of aberrant PI3K/Akt signaling by Salubrinal in a GalT-deficient mouse model.

Classic Galactosemia is an autosomal recessive disorder caused by deleterious mutations in the GALT gene, which encodes galactose-1 phosphate uridylyltransferase enzyme (GALT: EC 2.7.7.12). Recent studies of primary skin fibroblasts isolated from the GalT-deficient mice demonstrated a slower growth rate, a higher level of endoplasmic reticulum (ER) stress, and down-regulation of the Phosphoinositide 3 kinase/Protein kinase B (PI3K/Akt) signaling pathway. In this study, we compared the expression levels of the PI3K/Akt signaling pathway in normal and GalT-deficient mouse tissues. In mutant mouse ovaries, phospho-Akt [pAkt (Ser473)] and pGsk3ß were reduced by 62.5% and 93.5%, respectively (p<0.05 versus normal controls). In mutant cerebella, pAkt (Ser473) and pGsk3ß were reduced by 62%, 50%, respectively (p<0.05). To assess the role of ER stress in the down-regulation of PI3K/Akt signaling, we examined if administration of Salubrinal, a chemical compound that alleviates ER stress, to GalT-deficient fibroblasts and animals could normalize the pathway. Our results demonstrated that Salubrinal effectively reversed the down-regulated PI3K/Akt signaling pathway in the mutant cells and animals to levels close to those of their normal counterparts. Moreover, we revealed that Salubrinal can significantly slow down the loss of Purkinje cells in the cerebella, as well as the premature loss of primordial ovarian follicles in young mutant mice. These results open the door for a new therapeutic approach for the patients with Classic Galactosemia.

Classical Galactosemia: Insight into Molecular Pathomechanisms by Differential Membrane Proteomics of Fibroblasts under Galactose Stress.

Classical galactosemia, a hereditary metabolic disease caused by the deficiency of galactose-1-phosphate uridyltransferase (GALT; EC 2.7.712), results in an impaired galactose metabolism and serious long-term developmental affection of the CNS and ovaries, potentially related in part to endogenous galactose-induced protein dysglycosylation. In search for galactose-induced changes in membrane raft proteomes of GALT-deficient cells, we performed differential analyses of lipid rafts from patient-derived (Q) and sex- and age-matched control fibroblasts (H) in the presence or absence of the stressor. Label-based proteomics revealed of the total 454 (female) or 678 (male) proteins a proportion of ∼12% in at least one of four relevant ratios as fold-changed. GALT(-) cell-specific effects in the absence of stressor revealed cell-model-dependent affection of biological processes related to protein targeting to the plasma membrane (female) or to cellular migration (male). However, a series of common galactose-induced effects were observed, among them the strongly increased ER-stress marker GRP78 and calreticulin involved in N-glycoprotein quality control. The membrane-anchored N-glycoprotein receptor CD109 was concertedly decreased under galactose-stress together with cadherin-13, GLIPR1, glypican-1, and semaphorin-7A. A series of proteins showed opposite fold-changes in the two cell models, whereas others fluctuated in only one of the two models.

Insight into the mechanism of galactokinase: Role of a critical glutamate residue and helix/coil transitions.

Galactokinase, the enzyme which catalyses the first committed step in the Leloir pathway, has attracted interest due to its potential as a biocatalyst and as a possible drug target in the treatment of type I galactosemia. The mechanism of the enzyme is not fully elucidated. Molecular dynamics (MD) simulations of galactokinase with the active site residues Arg-37 and Asp-186 altered predicted that two regions (residues 174-179 and 231-240) had different dynamics as a consequence. Interestingly, the same two regions were also affected by alterations in Arg-105, Glu-174 and Arg-228. These three residues were identified as important in catalysis in previous computational studies on human galactokinase. Alteration of Arg-105 to methionine resulted in a modest reduction in activity with little change in stability. When Arg-228 was changed to methionine, the enzyme's interaction with both ATP and galactose was affected. This variant was significantly less stable than the wild-type protein. Changing Glu-174 to glutamine (but not to aspartate) resulted in no detectable activity and a less stable enzyme. Overall, these combined in silico and in vitro studies demonstrate the importance of a negative charge at position 174 and highlight the critical role of the dynamics in to key regions of the protein. We postulate that these regions may be critical for mediating the enzyme's structure and function.

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts.

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3ß (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.

Spontaneous Recovery of Ovarian Function in an Adolescent with Galactosemia and Apparent Premature Ovarian Insufficiency.

BACKGROUND: Galactosemia is an inborn error of metabolism resulting in premature ovarian insufficiency in 80-90% of females. There have been no reported cases of biochemical ovarian failure followed by normal menses. CASE: A 12-year-old girl with galactosemia presented for gynecologic consultation. Her follicle-stimulating hormone (FSH) and estradiol levels were 52.9 U/L and less than 100 pmol/L, respectively. She started exogenous estrogen to stimulate puberty. At 16, she had spontaneous regular menstrual cycles. FSH and luteinizing hormone (LH) levels reflected normal ovarian function. Hormonal contraception was provided. One year later, she was found to be in ovarian failure (FSH 86.6 U/L, LH 33.3 U/L), and both estradiol and anti-Müllerian hormone were undetectable. SUMMARY AND CONCLUSIONS: This case documents spontaneous resumption of ovarian function after galactosemia-related ovarian failure. The use of FSH and LH is potentially limited in predicting ovarian function in this population.

Effects of topically applied tocotrienol on cataractogenesis and lens redox status in galactosemic rats.

PURPOSE: Oxidative and nitrosative stress underlies cataractogenesis, and therefore, various antioxidants have been investigated for anticataract properties. Several vitamin E analogs have also been studied for anticataract effects due to their antioxidant properties; however, the anticataract properties of tocotrienols have not been investigated. In this study, we investigated the effects of topically applied tocotrienol on the onset and progression of cataract and lenticular oxidative and nitrosative stress in galactosemic rats. METHODS: In the first part of this study, we investigated the effects of topically applied microemulsion formulation of tocotrienol (TTE) using six concentrations ranging from 0.01% to 0.2%. Eight groups of Sprague-Dawley rats (n = 9) received distilled water, vehicle, or one of the six TTE concentrations as pretreatment topically twice daily for 3 weeks while on a normal diet. After pretreatment, animals in groups 2-8 received a 25% galactose diet whereas group 1 continued on the normal diet for 4 weeks. During this 4-week period, topical treatment continued as for pretreatment. Weekly slit-lamp examination was conducted to assess cataract progression. At the end of the experimental period, the animals were euthanized, and the proteins and oxidative stress parameters were estimated in the lenses. In the second part of the study, we compared the anticataract efficacy of the TTE with the liposomal formulation of tocotrienol (TTL) using five groups of Sprague-Dawley rats (n = 15) that received distilled water, TTE, TTL, or corresponding vehicle. The mode of administration and dosing schedule were the same as in study 1. Weekly ophthalmic examination and lens protein and oxidative stress estimates were performed as in study 1. Lens nitrosative stress was also estimated. RESULTS: During the 4-week treatment period, the groups treated with 0.03% and 0.02% tocotrienol showed slower progression of cataract compared to the vehicle-treated group (p<0.05), whereas the group treated with 0.2% tocotrienol showed faster progression of cataract compared to the vehicle-treated group (p<0.05). The lenticular protein content, malondialdehyde, superoxide dismutase, and catalase levels were normalized in the groups that received 0.03% and 0.02% tocotrienol. The lenticular reduced glutathione also showed a trend toward normalization in these groups. In contrast, the group treated with 0.2% tocotrienol showed increased lenticular oxidative stress. When the microemulsion and liposomal formulations were compared, the effects on cataract progression, lens oxidative and nitrosative stress, and lens protein content did not show significant differences. CONCLUSIONS: Topically applied tocotrienol within the concentration range of less than 0.05% and more than 0.01% tends to delay the onset and progression of cataract in galactose-fed rats by reducing lenticular oxidative and nitrosative stress. However, topical tocotrienol at a concentration of 0.2% and higher aggravates cataractogenesis in galactose-fed rats by increasing lens oxidative stress. The anticataract efficacy of 0.03% microemulsion of tocotrienol did not differ from its liposomal formulations at the same concentration.

Manganese-based superoxide dismutase mimics modify both acute and long-term outcome severity in a Drosophila melanogaster model of classic galactosemia.

AIMS: The goal of this study was to use two manganese (Mn)-based superoxide dismutase (SOD) mimics to test the hypothesis that reactive oxygen species contribute to both acute and long-term outcomes in a galactose-1P uridylyltransferase (GALT)-null Drosophila melanogaster model of classic galactosemia. RESULTS: We tested the impact of each of two Mn porphyrin SOD mimics, MnTnBuOE-2-PyP(5+), and MnTE-2-PyP(5+), (i) on survival of GALT-null Drosophila larvae reared in the presence versus absence of dietary galactose and (ii) on the severity of a long-term movement defect in GALT-null adult flies. Both SOD mimics conferred a significant survival benefit to GALT-null larvae exposed to galactose but not to controls or to GALT-null larvae reared in the absence of galactose. One mimic, MnTE-2-PyP(5+), also largely rescued a galactose-independent long-term movement defect otherwise seen in adult GALT-null flies. The survival benefit of both SOD mimics occurred despite continued accumulation of elevated galactose-1P in the treated animals, and studies of thiolated proteins demonstrated that in both the presence and absence of dietary galactose MnTE-2-PyP(5+) largely prevented the elevated protein oxidative damage otherwise seen in GALT-null animals relative to controls. INNOVATION AND CONCLUSIONS: Our results confirm oxidative stress as a mediator of acute galactose sensitivity in GALT-null Drosophila larvae and demonstrate for the first time that oxidative stress may also contribute to galactose-independent adult outcomes in GALT deficiency. Finally, our results demonstrate for the first time that both MnTnBuOE-2-PyP(5+) and MnTE-2-PyP(5+) are bioavailable and effective when administered through an oral route in a D. melanogaster model of classic galactosemia.

Oxidative stress contributes to outcome severity in a Drosophila melanogaster model of classic galactosemia.

Classic galactosemia is a genetic disorder that results from profound loss of galactose-1P-uridylyltransferase (GALT). Affected infants experience a rapid escalation of potentially lethal acute symptoms following exposure to milk. Dietary restriction of galactose prevents or resolves the acute sequelae; however, many patients experience profound long-term complications. Despite decades of research, the mechanisms that underlie pathophysiology in classic galactosemia remain unclear. Recently, we developed a Drosophila melanogaster model of classic galactosemia and demonstrated that, like patients, GALT-null Drosophila succumb in development if exposed to galactose but live if maintained on a galactose-restricted diet. Prior models of experimental galactosemia have implicated a possible association between galactose exposure and oxidative stress. Here we describe application of our fly genetic model of galactosemia to the question of whether oxidative stress contributes to the acute galactose sensitivity of GALT-null animals. Our first approach tested the impact of pro- and antioxidant food supplements on the survival of GALT-null and control larvae. We observed a clear pattern: the oxidants paraquat and DMSO each had a negative impact on the survival of mutant but not control animals exposed to galactose, and the antioxidants vitamin C and α-mangostin each had the opposite effect. Biochemical markers also confirmed that galactose and paraquat synergistically increased oxidative stress on all cohorts tested but, interestingly, the mutant animals showed a decreased response relative to controls. Finally, we tested the expression levels of two transcripts responsive to oxidative stress, GSTD6 and GSTE7, in mutant and control larvae exposed to galactose and found that both genes were induced, one by more than 40-fold. Combined, these results implicate oxidative stress and response as contributing factors in the acute galactose sensitivity of GALT-null Drosophila and, by extension, suggest that reactive oxygen species might also contribute to the acute pathophysiology in classic galactosemia.

Evidence of cataplerosis in a patient with neonatal classical galactosemia presenting as citrin deficiency.

Classical galactosemia is an autosomal recessive disorder caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase. Undoubtedly, some of the short term complications are linked to the toxic effects of the accumulated abnormal metabolites (galactose-1-phosphate and galactitol). However, the physiopathology of neonatal liver failure remains unclear. We report the case of a 7-week-old girl who was first diagnosed with liver failure, hypoprotidaemia, ascites and generalized edemas. High citrulline (293 micromol/L), on initial plasma amino acid, suggested the diagnosis of citrin deficiency. As the citric acid cycle intermediates were non-detectable (oxoglutarate, succinate and citrate), a cataplerotic state was suspected. As a result, citrate (as an anaplerotic treatment) induced a clear improvement in her liver function. Four weeks later, this patient was switched to a galactose-free formula (as recommended in citrin deficiency with galactosemia) and her pathological status returned to normal. Citrin deficiency was later ruled out by molecular biology studies; then we reintroduced a galactose-containing formula which re-evoked rapidly vomiting, galactose aversion and hepatic cytolysis and the diagnosis of classical galactosemia was established. Our case clearly shows that cataplerosis could play a role in the pathophysiology of the neonatal liver disease observed in classical galactosemia.

Akt activation and augmented fibronectin production in hyperhexosemia.

Dysmetabolic state in diabetes may lead to augmented synthesis of extracellular matrix (ECM) proteins. In the endothelial cells, we have previously demonstrated that glucose-induced fibronectin (FN) production and that of its splice variant, EDB(+)FN, is regulated by protein kinase B (PKB, also known as Akt). In this study, we investigated the role of Akt1 in ECM protein production in the organs affected by chronic diabetic complications. We studied Akt1/PKBalpha knockout mice and wild-type control littermates. To avoid confounding effects of systemic insulin, we used 30% galactose feeding to induce hyperhexosemia for 8 wk starting at 6 wk of age. We investigated FN mRNA, EDB(+)FN mRNA, and transforming growth factor (TGF)-beta mRNA expression, Akt phosphorylation, Akt kinase activity, and NF-kappaB and AP-1 activation in the retina, heart, and kidney. Renal and cardiac tissues were histologically examined. Galactose feeding caused significant upregulation of FN, EDB(+)FN, and TGF-beta in all tissues. FN protein levels paralleled mRNA. Such upregulation were prevented in Akt1-deficient galactose-fed mice. Galactose feeding caused ECM protein deposition in the glomeruli and in the myocardium, which was prevented in the Akt knockout mice. NF-kappaB and AP-1 activation was pronounced in galactose-fed wild-type mice and prevented in the galactose-fed Akt1/PKBalpha-deficient group. In the retina and kidney, Ser473 was the predominant site for Akt phosphorylation, whereas in the heart it was Thr308. Parallel experiment in streptozotocin-induced diabetic animals showed similar results. The data from this study indicate that hyperhexosemia-induced Akt/PKB activation may be an important mechanism leading to NF-kappaB and AP-1 activation and increased ECM protein synthesis in the organs affected by chronic diabetic complications.

The unfolded protein response in lens epithelial cells from galactosemic rat lenses.

PURPOSE: Diabetic complications are associated with hypoglycemia and hyperglycemia. The purpose of this study was to investigate the effect of both glucose deprivation and hyperglycemia on the induction of endoplasmic reticulum (ER) stress and the subsequent activation of the unfolded protein response (UPR) that results in apoptosis in in vitro cultured lens epithelial cells (LECs) and in vivo cataract formation in galactose-fed rats. METHODS: Lenses from rats fed a standard diet containing 50% galactose with or without an aldose reductase inhibitor (ARI) were investigated. Transformed human LECs were cultured in standard 10% FCS-DMEM containing various concentrations of sugar. UPR-specific proteins from both the rat lenses and lens cultures were quantified by protein blot analysis. Cell death was evaluated with TUNEL staining and ethidium homodimer-1 (EthD) dyes. Reactive oxygen species (ROS) were quantified with H2-DCF, and free glutathione (GSH) levels were measured with a commercial GSH quantification kit. RESULTS: Increased apoptosis of the LECs was observed in the lenses of rats fed the galactose diet for 5 to 9 days, and nuclear cataracts subsequently developed in these lenses after 13 to 15 days. Protein blot analysis of the LECs from these galactose-fed rats showed higher levels of the UPR-specific proteins Bip/GRP78, ATF4, and CHOP. These LECs also demonstrated activation of the UPR-specific procaspase-12 and the increased presence of ROS, whereas GSH was reduced. Because these results indicate that the UPR is activated in LECs along with the production of ROS and apoptosis during cataract formation in the galactose-fed rats, subsequent studies were conducted to determine the role of nonenzymatic glycation, osmotic stress, and oxidative stress on these biochemical processes. In vitro cultures of human LECs showed that the UPR was induced by osmotic and oxidative stress, but not by glycation. In addition, the UPR and apoptosis in LECs was induced by glucose deprivation. The ARI blocked the induction of the UPR, cell death, and cataract formation. CONCLUSIONS: The UPR that is induced by abnormally high or low concentrations of sugar is linked to the production of ROS, increased apoptosis in LECs, and cataract formation. The inhibition of the UPR induction by ARI suggests that osmotic stress may be the primary inducer of the UPR. Modulation of the UPR pathways may offer novel methods for the development of therapeutic tools to delay cataracts.

Suckling rat brain regional distribution of Na+,K+-atpase activity in the in vitro galactosaemia: the effect of L-cysteine and glutathione.

Inhibition of Na+,K+-ATPase activity causes edema and cell death in central nervous system. We determined the in vitro effects of galactose-l-phosphate (Gal-1-P), galactitol (Galtol) and galactose (Gal) (mix A = classical galactosaemia) or Galtol and Gal (mix B = galactokinase deficiency galactosaemia), on Na+,K+-ATPase activity in suckling rat brain frontal cortex, hippocampus or hypothalamus homogenates. Gal-1-P or Galtol alone at different concentrations, significantly inhibited Na+,K+-ATPase whereas Gal activated the enzyme in all investigated brain regions. Both mix A and mix B inactivated the enzyme by 20-30% (p < 0.001) in all studied areas. L-Cysteine (Cys) and glutathione (GSH) supplementation in mix B not only reversed the enzyme inhibition but also resulted in an activation of 50-60%, (p < 0.001) in all brain areas. Their presence in mix A also activated the inhibited Na+,K+-ATPase in hippocampus and hypothalamus to a lower degree, whereas Cys reversed the frontal cortex enzyme activity to control value only. These findings indicate that oxidation of the enzyme critical groups may be involved in galactosaemia, producing inhibitory effect. This phenomenon is reversed by antioxidants Cys and GSH, implying that free radicals may be implicated in the observed enzyme inactivation.

The protective effect of L-cysteine and glutathione on the adult and aged rat brain (Na+,K+)-ATPase and Mg2+-ATPase activities in galactosemia in vitro.

The aim of this study was to evaluate whether the addition of the antioxidants L-cysteine (Cys) or the reduced glutathione (GSH) could reverse the alterations of brain total antioxidant status (TAS) and the modulated activities of the enzymes (Na+,K+)-ATPase, and Mg2+-ATPase in adult or aged rat brain homogenates induced by galactosemia in vitro. Mixture A [mix. A: galactose-1-phosphate (Gal-1-P, 2 mM) plus galactitol (Galtol, 2 mM) plus galactose (Gal, 4 mM) = classical galactosemia] or mixture B [mix. B: Galtol (2 mM) plus Gal (1 mM) = galactokinase deficiency galactosemia] were preincubated in the presence or absence of Cys (0.83 mM) or GSH (0.83 mM) with adult or aged brain homogenates at 37 degrees C for 1 h. TAS and the enzyme activities were determined spectrophotometrically. Mix. A or mix. B preincubation with the adult brain resulted in a significant (Na+,K+)-ATPase inhibition (-30%) and a Mg2+-ATPase stimulation (+300% and +33%, respectively), whereas lower modifications of the enzyme activities (p < 0.001) were found in the aged brain. Gal mixtures decreased TAS by 40% (p < 0.001) and by 20% (p < 0.01) in adult and aged samples, respectively. The antioxidants significantly increased TAS resulting in the reversion of (Na+,K+)-ATPase inhibition and Mg2+-ATPase stimulation by mix. B only. The inhibitory effect of Gal and its derivatives on brain (Na+,K+)-ATPase and their stimulatory effect on Mg2+-ATPase are being decreased with age, probably due to the producion of free radicals. Cys and GSH increased TAS resulting in a reversion of the inhibited (Na+,K+)-ATPase in both models of the in vitro galactosemia and the stimulated Mg2+-ATPase in galactokinase deficiency galactosemia only.

The effect of diet on total antioxidant status, erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in patients with classical galactosaemia.

OBJECTIVE: Classical galactosaemia is characterized by high levels of galactose-1-phosphate (Gal-1-P), galactose and galactitol. In vitro studies have shown modulation of the rat brain Na+,K+-ATPase and Mg2+-ATPase activities by Gal-1-P. The aim of this study was to evaluate the erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase activities in galactosaemic patients and to correlate them to Gal-1-P, total antioxidant status (TAS) and membrane protein content (PC). PATIENTS AND METHODS: Nine patients (N=9) originally on "loose diet" (group B) were requested to follow their diet strictly (group A). Twelve healthy children were the controls (group C). The activities of the enzymes, TAS and Gal-1-P in blood were determined spectrophotometrically. In the in vitro study, erythrocyte membranes from controls were preincubated with Gal-1-P (300 microM), and then with l-cysteine (0.83 mM) or reduced glutathione (0.83 mM) whereas these from the patients with the antioxidants only. RESULTS: Na+,K+-ATPase, Mg2+-ATPase, TAS and PC were significantly (P<0.001) reduced (0.31+/-0.03, 1.7+/-0.2 micromol Pi/hxmg protein, 0.89+/-0.02 mmol/l, 36.8+/-2.0 g/l, respectively) in group B as compared with those of group A (0.58+/-0.06, 2.5+/-0.2 micromol Pi/hxmg protein, 1.41+/-0.11 mmol/l, 51.5+/-3.1g/l, respectively) and controls (0.67+/-0.05, 3.2+/-0.2 micromol Pi/hxmg protein, 1.65+/-0.12 mmol/l, 64.0+/-3.5 g/l, respectively). Gal-1-P levels in group B was significantly higher than those in group A and controls. Positive correlation coefficients were found between the enzyme activities, PC and TAS whereas Gal-1-P inversely correlated to the enzyme activities. Incubation of the erythrocyte membranes from the patients with the antioxidants failed to restore the activities of inhibited enzymes, whereas the inhibition by Gal-1-P in controls was reversed. CONCLUSIONS: High blood Gal-1-P concentrations resulted in low TAS and PC. The inhibition of Na+,K+-ATPase and Mg2+-ATPase may be due to the presence of free radicals and/or the elevated Gal-1-P.