Galactose-1-phosphate uridyltransferase deficiency: A literature review of the putative mechanisms of short and long-term complications and allelic variants.

Galactosemia type 1 is an autosomal recessive disorder of galactose metabolism, determined by a deficiency in the enzyme galactose-1-phosphate uridyltransferase (GALT). GALT deficiency is classified as severe or variant depending on biochemical phenotype, genotype and potential to develop acute and long-term complications. Neonatal symptoms usually resolve after galactose-restricted diet; however, some patients, despite the diet, can develop long-term complications, in particular when the GALT enzyme activity results absent or severely decreased. The mechanisms of acute and long-term complications are still discussed and several hypotheses are presented in the literature like enzymatic inhibition, osmotic stress, endoplasmic reticulum stress, oxidative stress, defects of glycosylation or epigenetic modification. This review summarizes the current knowledge of galactosemia, in particular the putative mechanisms of neonatal and long-term complications and the molecular genetics of GALT deficiency.

Ochratoxin A as possible factor trigging autism and its male prevalence via epigenetic mechanism.

The role of dysbiosis causing leaky gut with xenobiotic production and absorption is increasingly demonstrated in autism spectrum disorder (ASD) pathogenesis. Among xenobiotics, we focused on ochratoxin A (one of the major food contaminating mycotoxin), that in vitro and in vivo exerts a male-specific neurotoxicity probably via microRNA modulation of a specific target gene. Among possible targets, we focused on neuroligin4X. Interestingly, this gene carries some single nucleotide polymorphisms (SNPs) already correlated with the disease and with illegitimate microRNA binding sites and, being located on X-chromosome, could explain the male prevalence. In conclusion, we propose a possible gene-environment interaction triggering ASD explaining the epigenetic neurotoxic mechanism activated by ochratoxin A in genetically predisposed children. This mechanism offers a clue for male prevalence of the disease and may have an important impact on prevention and cure of ASD.

4-Nonylphenol reduces cell viability and induces apoptosis and ER-stress in a human epithelial intestinal cell line.

4-Nonylphenol is a widely diffused and stable environmental contaminant, originating from the degradation of alkyl phenol ethoxylates, common surfactants employed in several industrial applications. Due to its hydrophobic nature, 4-nonylphenol can easily accumulate in living organisms, including humans, where it displays a wide range of toxic effects. Since the gastrointestinal tract represents the main route by which 4-nonylphenol enters the body, the intestine may be one of the first organs to be damaged by chronic exposure to this pollutant through the diet. In the present study, we investigated the effects of 4-nonylphenol on a human intestinal epithelial cell line (Caco-2 cells). We demonstrated that 4-nonylphenol was cytotoxic to cells, as revealed by a decrease of the cell number and the decrement of mitochondrial functionality after 24 h of treatment. 4-Nonylphenol also reduced the number of cells entering into S-phase and interfered with epidermal growth factor signalling, with consequent negative effects on cell survival. In addition, 4-nonylphenol induced apoptosis, involving the activation of caspase-3, and triggered an endoplasmic reticulum-stress response, as revealed by over-expression of GRP78 (78 kDa glucose-regulated protein) and activation of XBP1 (X-box binding protein-1). Together, these findings support the hypothesis that prolonged exposure to 4-nonylphenol through the diet may lead to local damage at the level of intestinal mucosa, with potentially negative consequences for intestinal homeostasis and functionality.

Clinical and molecular spectra in galactosemic patients from neonatal screening in northeastern Italy: structural and functional characterization of new variations in the galactose-1-phosphate uridyltransferase (GALT) gene.

Classical galactosemia is an autosomal recessive inborn error of metabolism due to mutations of the GALT gene leading to toxic accumulation of galactose and derived metabolites. With the benefit of early diagnosis by neonatal screening and early therapy, the acute presentation of classical galactosemia can be prevented. However, despite early diagnosis and treatment, the long term outcome for these patients is still unpredictable because they may go on to develop cognitive disability, speech problems, neurological and/or movement disorders and, in females, ovarian dysfunction. The objectives of the current study were to report our experience with a group of galactosemic patients identified through the neonatal screening programs in northeastern Italy during the last 30years. No neonatal deaths due to galactosemia complications occurred after the introduction of the neonatal screening program. However, despite the early diagnosis and dietary treatment, the patients with classical galactosemia showed one or more long-term complications. A total of 18 different variations in the GALT gene were found in the patient cohort: 12 missense, 2 frameshift, 1 nonsense, 1 deletion, 1 silent variation, and 1 intronic. Six (p.R33P, p.G83V, p.P244S, p.L267R, p.L267V, p.E271D) were new variations. The most common variation was p.Q188R (12 alleles, 31.5%), followed by p.K285N (6 alleles, 15.7%) and p.N314D (6 alleles, 15.7%). The other variations comprised 1 or 2 alleles. In the patients carrying a new mutation, the biochemical analysis of GALT activity in erythrocytes showed an activity of <1%. In silico analysis (SIFT, PolyPhen-2 and the computational analysis on the static protein structure) showed potentially damaging effects of the six new variations on the GALT protein, thus expanding the genetic spectrum of GALT variations in Italy. The study emphasizes the difficulty in establishing a genotype-phenotype correlation in classical galactosemia and underlines the importance of molecular diagnostic testing prior to making any treatment.

Correlation between fluorescence and structure in the orange-emitting GFP-like protein, monomeric Kusabira Orange.

The mKO is the monomeric version of Kusabira Orange, a GFP-like protein emitting bright orange fluorescence at 559 nm. This protein shows the characteristic ß-barrel motif typical of the fluorescent protein family which it belongs to, similar spectral properties to the tetrameric form and an exceptional photo-stability to pH changes. Here, we demonstrate that mKO in solution at physiological pH exhibits a secondary structure analogue to that of the crystal. Moreover, we describe the thermal unfolding, revealing an outstanding structural stability with a denaturation temperature close to 90 °C and identifying the existence of a thermodynamic intermediate. The denaturation process of mKO results to be absolutely irreversible because of the complete lost of the native structure and the consequent aggregation, while the presence of the intermediate state is most likely due to coexistence of two different species of mKO, with protonated and deprotonated chromophore respectively, that affects the fluorescence properties and the structural stability of the protein.

Myosin-binding protein C DNA variants in domestic cats (A31P, A74T, R820W) and their association with hypertrophic cardiomyopathy.

BACKGROUND: Two mutations in the MYBPC3 gene have been identified in Maine Coon (MCO) and Ragdoll (RD) cats with hypertrophic cardiomyopathy (HCM). OBJECTIVE: This study examined the frequency of these mutations and of the A74T polymorphism to describe their worldwide distribution and correlation with echocardiography. ANIMALS: 1855 cats representing 28 breeds and random-bred cats worldwide, of which 446 underwent echocardiographic examination. METHODS: This is a prospective cross-sectional study. Polymorphisms were genotyped by Illumina VeraCode GoldenGate or by direct sequencing. The disease status was defined by echocardiography according to established guidelines. Odds ratios for the joint probability of having HCM and the alleles were calculated by meta-analysis. Functional analysis was simulated. RESULTS: The MYBPC3 A31P and R820W were restricted to MCO and RD, respectively. Both purebred and random-bred cats had HCM and the incidence increased with age. The A74T polymorphism was not associated with any phenotype. HCM was most prevalent in MCO homozygote for the A31P mutation and the penetrance increased with age. The penetrance of the heterozygote genotype was lower (0.08) compared with the P/P genotype (0.58) in MCO. CONCLUSIONS AND CLINICAL IMPORTANCE: A31P mutation occurs frequently in MCO cats. The high incidence of HCM in homozygotes for the mutation supports the causal nature of the A31P mutation. Penetrance is incomplete for heterozygotes at A31P locus, at least at a young age. The A74T variant does not appear to be correlated with HCM.

Analysis of galactosemia-linked mutations of GALT enzyme using a computational biology approach.

We describe the prediction of the structural and functional effects of mutations on the enzyme galactose-1-phosphate uridyltransferase related to the genetic disease galactosemia, using a fully computational approach. One hundred and seven single-point mutants were simulated starting from the structural model of the enzyme obtained by homology modeling methods. Several bioinformatics programs were then applied to each resulting mutant protein to analyze the effect of the mutations. The mutations have a direct effect on the active site, or on the dimer assembly and stability, or on the monomer stability. We describe how mutations may exert their effect at a molecular level by altering H-bonds, salt bridges, secondary structure or surface features. The alteration of protein stability, at level of monomer and/or dimer, is the main effect observed. We found an agreement between our results and the functional experimental data available in literature for some mutants. The data and analyses for all the mutants are fully available in the web-accessible database hosted at http://bioinformatica.isa.cnr.it/GALT.

Photorefractive keratectomy in high myopic defects with or without intraoperative mitomycin C: 1-year results.

PURPOSE: To study the results of the prophylactic use of mitomycin C (MMC) to reduce haze formation and refractive regression after excimer laser photorefractive keratectomy (PRK) for high myopic defects (>5 diopters). METHODS: Prospective, consecutive, observational study. A total of 124 eyes of 62 patients were divided into two groups of 31 patients, 62 eyes each (Groups A and B). Only Group A was treated with MMC 0.02%. The data of the two groups of eyes, related to the best-corrected visual acuity (BCVA), to the difference of refraction pre- and post-treatment, and to the corneal haze, were analyzed through combined permutation tests by using the NPC Test software . RESULTS: BCVA of Group A, 1 year after treatment, was better than that of the control Group B (one-sided p value = 0.013): Group A - 3 eyes (4.8%) had a loss of a decimal fraction and no eyes > 1; Group B - 13 eyes (20.9%) had a loss of a decimal fraction and 1 eye (1.6%) of 2. There was a smaller difference between attempted and achieved SE correction in Group A with respect to Group B (one-sided p value = 0.068): Group A - 43 eyes (69.3%) within +/- 0.50 D; Group B - 31 eyes (50%) within +/- 0.50 D. there was a smaller incidence of corneal haze in the group for which MMC was used (one-sided p value = 0.005). CONCLUSIONS: In this study, the application of MMC 0.02% solution immediately after PRK produced lower haze rates and had better predictability and improved efficacy 1 year after treatment.

Temporary graft for closed-system cataract surgery during corneal triple procedures.

Performing phacoemulsification during a triple corneal procedure has many advantages. Operating in a closed chamber makes surgery easier and safer. In some cases, however, a dense corneal opacity may prevent closed-chamber surgery, necessitating the use of an open-sky technique. In these cases, a temporary corneal graft using a corneal button not suitable for penetrating keratoplasty is proposed to allow phacoemulsification and foldable intraocular lens implantation through a corneal tunnel. The temporary corneal graft is replaced with a permanent graft after these steps are completed. This technique was effective in 3 patients with cataract and dense corneal opacity.

Involutional entropion surgery: a modified technique.

PURPOSE: A study to evaluate the effectiveness of a modified procedure to correct involutional entropion. METHODS: Seventeen cases of involutional entropion underwent surgery and had a postoperative follow-up of 18 months. The authors describe a technique of vertically shortening the anterior lamella (skin and orbicularis muscle), using a blepharoplasty incision and reflection of a skin muscle flap. RESULTS: In 16 of 17 eyelids affected by involutional entropion, this operative procedure showed good functional outcome (good correction of the relation between lower eyelid edge and eyeball) and aesthetic outcome (no hypertrophic scar, dyschromia or unnatural folding of the skin). CONCLUSIONS: A correct approach to entropion surgery needs an accurate preoperative evaluation of the individual physiopathogenic factors. This procedure gives lasting functional and pleasing cosmetic results when preseptal orbicularis muscle override has been identified as the cause of senile entropion.

Allosteric communication of tryptophan synthase. Functional and regulatory properties of the beta S178P mutant.

The alpha(2)beta(2) tryptophan synthase complex is a model enzyme for understanding allosteric regulation. We report the functional and regulatory properties of the betaS178P mutant. Ser-178 is located at the end of helix 6 of the beta subunit, belonging to the domain involved in intersubunit signaling. The carbonyl group of betaSer-178 is hydrogen bonded to Gly-181 of loop 6 of the alpha subunit only when alpha subunit ligands are bound. An analysis by molecular modeling of the structural effects caused by the betaS178P mutation suggests that the hydrogen bond involving alphaGly-181 is disrupted as a result of localized structural perturbations. The ratio of alpha to beta subunit concentrations was calculated to be 0.7, as for the wild type, indicating the maintenance of a tight alpha-beta complex. Both the activity of the alpha subunit and the inhibitory effect of the alpha subunit ligands indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate were found to be the same for the mutant and wild type enzyme, whereas the beta subunit activity of the mutant exhibited a 2-fold decrease. In striking contrast to that observed for the wild type, the allosteric effectors indole-3-acetylglycine and d,l-alpha-glycerol-3-phosphate do not affect the beta activity. Accordingly, the distribution of l-serine intermediates at the beta-site, dominated by the alpha-aminoacrylate, is only slightly influenced by alpha subunit ligands. Binding of sodium ions is weaker in the mutant than in the wild type and leads to a limited increase of the amount of the external aldimine intermediate, even at high pH, whereas binding of cesium ions exhibits the same affinity and effects as in the wild type, leading to an increase of the alpha-aminoacrylate tautomer absorbing at 450 nm. Crystals of the betaS178P mutant were grown, and their functional and regulatory properties were investigated by polarized absorption microspectrophotometry. These findings indicate that (i) the reciprocal activation of the alpha and beta activity in the alpha2beta2 complex with respect to the isolated subunits results from interactions that involve residues different from betaSer-178 and (ii) betaSer-178 is a critical residue in ligand-triggered signals between alpha and beta active sites.

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands.

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.

Novel allosteric effectors of the tryptophan synthase alpha(2)beta(2) complex identified by computer-assisted molecular modeling.

Tryptophan synthase is a pyridoxal 5'-phosphate-dependent alpha(2)beta(2) complex catalyzing the formation of L-tryptophan. The functional properties of one subunit are allosterically regulated by ligands of the other subunit. Molecules tailored for binding to the alpha-active site were designed using as a starting model the three-dimensional structure of the complex between the enzyme from Salmonella typhimurium and the substrate analog indole-3-propanol phosphate. On the basis of molecular dynamics simulations, indole-3-acetyl-X, where X is glycine, alanine, valine and aspartate, and a few other structurally related compounds were found to be good candidates for ligands of the alpha-subunit. The binding of the designed compounds to the alpha-active site was evaluated by measuring the inhibition of the alpha-reaction of the enzyme from Salmonella typhimurium. The inhibition constants were found to vary between 0.3 and 1.7 mM. These alpha-subunit ligands do not bind to the beta-subunit, as indicated by the absence of effects on the rate of the beta-reaction in the isolated beta(2) dimer. A small inhibitory effect on the activity of the alpha(2)beta(2) complex was caused by indole-3-acetyl-glycine and indole-3-acetyl-aspartate whereas a small stimulatory effect was caused by indole-3-acetamide. Furthermore, indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide perturb the equilibrium of the catalytic intermediates formed at the beta-active site, stabilizing the alpha-aminoacrylate Schiff base. These results indicate that (i) indole-3-acetyl-glycine, indole-3-acetyl-aspartate and indole-3-acetamide bind to the alpha-subunit and act as allosteric effectors whereas indole-3-acetyl-valine and indole-3-acetyl-alanine only bind to the alpha-subunit, and (ii) the terminal phosphate present in the already known allosteric effectors of tryptophan synthase is not strictly required for the transmission of regulatory signals.

Reliability of standardized echography before vitreoretinal surgery for proliferative diabetic retinopathy.

Standardized A- and B-scan echography was performed in 45 patients (68 eyes) affected by proliferative diabetic retinopathy, before vitreoretinal surgery, to evaluate the percentage of agreement between the ultrasonographic data and the surgical findings. The agreement was quite good for tractional retinal detachment (93%) and for posterior vitreous detachment (91%). The main cause of failure was the presence of vitreoschisis, while preretinal and vitreal membranes can easily be detected.

Echographic study of the vitreoretinal interface in giant retinal tears.

Vitreous and vitreoretinal interface changes were evaluated in giant retinal tears in order to plan surgical approach. Vitreal sineresis was evident in patients affected by primary giant retinal tears; in patients affected by retinal disinsertion the vitreous was adherent to the edge of the posterior flap, and in giant retinal tears secondary to IOL implantation, posterior vitreous detachment but no sineresis was displayed.