Jasmonates and Ethylene Shape Floridoside Synthesis during Carposporogenesis in the Red Seaweed Grateloupia imbricata.

Floridoside is a galactosyl-glycerol compound that acts to supply UDP-galactose and functions as an organic osmolyte in response to salinity in Rhodophyta. Significantly, the UDP-galactose pool is shared for sulfated cell wall galactan synthesis, and, in turn, affected by thallus development alongside carposporogenesis induced by volatile growth regulators, such as ethylene and methyl jasmonate, in the red seaweed Grateloupia imbricata. In this study, we monitored changes in the floridoside reservoir through gene expression controlling both the galactose pool and glyceride pool under different reproductive stages of G. imbricata and we considered changing salinity conditions. Floridoside synthesis was followed by expression analysis of galactose-1-phosphate uridyltransferase (GALT) as UDP-galactose is obtained from UDP-glucose and glucose-1P, and through α-galactosidase gene expression as degradation of floridoside occurs through the cleavage of galactosyl residues. Meanwhile, glycerol 3-phosphate is connected with the galactoglyceride biosynthetic pathway by glycerol 3-phosphate dehydrogenase (G3PD), monogalactosyl diacylglyceride synthase (MGDGS), and digalactosyl diacylglyceride synthase (DGDGS). The results of our study confirm that low GALT transcripts are correlated with thalli softness to locate reproductive structures, as well as constricting the synthesis of UDP-hexoses for galactan backbone synthesis in the presence of two volatile regulators and methionine. Meanwhile, α-galactosidase modulates expression according to cystocarp maturation, and we found high transcripts in late development stages, as occurred in the presence of methyljasmonate, compared to early stages in ethylene. Regarding the acylglyceride pool, the upregulation of G3PD, MGDGS, and DGDGS gene expression in G. imbricata treated with MEJA supports lipid remodeling, as high levels of transcripts for MGDGS and DGDGS provide membrane stability during late development stages of cystocarps. Similar behavior is assumed in three naturally collected thalli development stages-namely, fertile, fertilized, and fertile-under 65 psu salinity conditions. Low transcripts for α-galactosidase and high for G3PD are reported in infertile and fertilized thalli, which is the opposite to high transcripts for α-galactosidase and low for G3PD encountered in fertile thalli within visible cystocarps compared to each of their corresponding stages in 35 psu. No significant changes are reported for MGDGS and DGDGS. It is concluded that cystocarp and thallus development stages affect galactose and glycerides pools with interwoven effects on cell wall polysaccharides.

Overexpression of UDP-sugar pyrophosphorylase leads to higher sensitivity towards galactose, providing new insights into the mechanisms of galactose toxicity in plants.

Galactose toxicity (Gal-Tox) is a widespread phenomenon ranging from Escherichia coli to mammals and plants. In plants, the predominant pathway for the conversion of galactose into UDP-galactose (UDP-Gal) and UDP-glucose is catalyzed by the enzymes galactokinase, UDP-sugar pyrophosphorylase (USP) and UDP-galactose 4-epimerase. Galactose is a major component of cell wall polymers, glycolipids and glycoproteins; therefore, it becomes surprising that exogenous addition of galactose leads to drastic root phenotypes including cessation of primary root growth and induction of lateral root formation. Currently, little is known about galactose-mediated toxicity in plants. In this study, we investigated the role of galactose-containing metabolites like galactose-1-phosphate (Gal-1P) and UDP-Gal in Gal-Tox. Recently published data from mouse models suggest that a reduction of the Gal-1P level via an mRNA-based therapy helps to overcome Gal-Tox. To test this hypothesis in plants, we created Arabidopsis thaliana lines overexpressing USP from Pisum sativum. USP enzyme assays confirmed a threefold higher enzyme activity in the overexpression lines leading to a significant reduction of the Gal-1P level in roots. Interestingly, the overexpression lines are phenotypically more sensitive to the exogenous addition of galactose (0.5 mmol L-1 Gal). Nucleotide sugar analysis via high-performance liquid chromatography-mass spectrometry revealed highly elevated UDP-Gal levels in roots of seedlings grown on 1.5 mmol L-1 galactose versus 1.5 mmol L-1 sucrose. Analysis of plant cell wall glycans by comprehensive microarray polymer profiling showed a high abundance of antibody binding recognizing arabinogalactanproteins and extensins under Gal-feeding conditions, indicating that glycoproteins are a major target for elevated UDP-Gal levels in plants.

Novel GALT variations and genetic spectrum in Turkish population with the correlation of genotype and phenotype.

Classic galactosemia (OMIM#230400) is an autosomal recessive inborn error of carbohydrate metabolism caused by a deficiency of the galactose-1-phosphate-uridyl-transferase enzyme encoded by the GALT gene. Even though a galactose-restricted diet efficiently resolves the acute complications, it is insufficient to prevent long-term complications regarding speech defects, intellectual functioning, premature ovarian failure, cataract, hepatomegaly, dysarthria, ataxia, and tremor. Seventy-seven patients who were genetically diagnosed with classic galactosemia were included in this cohort. Identified novel variants were classified based on their predicted effect on the GALT function. Further, potential genotype-phenotype correlations were investigated via statistical analysis. In total, 18 different sequence variants were identified, including four novels (c.200delG/p.(Arg67Profs* 19), c.533T>G/ p.(Met178Arg), c.708_709delGT/p.(Ser236Argfs* 30), c.467C>A/p.(Ser156* )). Jaundice was the most common short-term finding with 80% (61/77). Even with early diagnosis, intellectual disability is encountered with 36% (27/74) of the long-term complications. Patients with biallelic missense variants have a significantly higher prevalence of cataracts (OR: 17.9). Longitudinal observations showed attenuation of cataracts and hepatomegaly. This study has shown the GALT variation spectrum of the Turkish population with a 30-year retrospective cohort, submitting a significant contribution to the genotype/phenotype correlation in galactosemia. This study also highlights the cost-effective importance of Sanger sequencing in the diagnosis of single-gene metabolic diseases.

Addition of galactose-1-phosphate measurement enhances newborn screening for classical galactosemia.

Galactosemia is an inborn disorder of carbohydrate metabolism of which early detection can prevent severe illness. Although the assay for galactose-1-phosphate uridyltransferase (GALT) enzyme activity has been available since the 1960s, many issues prevented it from becoming universal. In order to develop the Israeli newborn screening pilot algorithm for galactosemia, flow injection analysis tandem mass spectrometry measurement of galactose-1-phosphate in archived dried blood spots from newborns with classical galactosemia, galactosemia variants, epimerase deficiency, and normal controls, was conducted. Out of 431 330 newborns screened during the pilot study (30 months), two with classical galactosemia and four with epimerase deficiency were identified and confirmed. Five false positives and no false negatives were recorded. Following this pilot study, the Israeli final and routine newborn screening algorithm, as recommended by the Advisory Committee to the National Newborn Screening Program, now consists of galactose-1-phosphate measurement integrated into the routine tandem mass spectrometry panel as the first-tier screening test, and GALT enzyme activity as the second-tier performed to identify only newborns suspected to be at risk for classical galactosemia. The GALT enzyme activity cut-off used in the final algorithm was lowered in order to avoid false positives.

Multi-omics in classical galactosemia: Evidence for the involvement of multiple metabolic pathways.

Classical galactosemia (CG) is one of the more frequent inborn errors of metabolism affecting approximately 1:40.000 people. Despite a life-saving galactose-restricted diet, patients develop highly variable long-term complications including intellectual disability and movement disorders. The pathophysiology of these complications is still poorly understood and development of new therapies is hampered by a lack of valid prognostic biomarkers. Multi-omics approaches may discover new biomarkers and improve prediction of patient outcome. In the current study, (semi-)targeted mass-spectrometry based metabolomics and lipidomics were performed in erythrocytes of 40 patients with both classical and variant phenotypes and 39 controls. Lipidomics did not show any significant changes or deficiencies. The metabolomics analysis revealed that CG does not only compromise the Leloir pathway, but also involves other metabolic pathways including glycolysis, the pentose phosphate pathway, and nucleotide metabolism in the erythrocyte. Moreover, the energy status of the cell appears to be compromised, with significantly decreased levels of ATP and ADP. This possibly is the consequence of two different mechanisms: impaired formation of ATP from ADP possibly due to reduced flux though the glycolytic pathway and trapping of phosphate in galactose-1-phosphate (Gal-1P) which accumulates in CG. Our findings are in line with the current notion that the accumulation of Gal-1P plays a key role in the pathophysiology of CG not only by depletion of intracellular phosphate levels but also by decreasing metabolite abundance downstream in the glycolytic pathway and affecting other pathways. New therapeutic options for CG could be directed towards the restoration of intracellular phosphate homeostasis.

S-Assimilation Influences in Carrageenan Biosynthesis Genes during Ethylene-Induced Carposporogenesis in Red Seaweed Grateloupia imbricata.

The synthesis of cell-wall sulfated galactans proceeds through UDP galactose, a major nucleotide sugar in red seaweed, whilst sulfate is transported through S-transporters into algae. Moreover, synthesis of ethylene, a volatile plant growth regulator that plays an important role in red seaweed reproduction, occurs through S-adenosyl methionine. This means that sulfur metabolism is involved in reproduction events as well as sulfated galactan synthesis of red seaweed. In this work we study the effects of methionine and MgSO4 on gene expression of polygalactan synthesis through phosphoglucomutase (PGM) and galactose 1 phosphate uridyltransferase (GALT) and of sulfate assimilation (S-transporter and sulfate adenylyltransferase, SAT) using treatment of ethylene for 15 min, which elicited cystocarp development in Grateloupia imbricata. Also, expressions of carbohydrate sulfotransferase and galactose-6-sulfurylase in charge of the addition and removal of sulfate groups to galactans backbone were examined. Outstanding results occurred in the presence of methionine, which provoked an increment in transcript number of genes encoding S-transporter and assimilation compared to controls regardless of the development stage of thalli. Otherwise, methionine diminished the transcript levels of PGM and GALT and expressions are associated with the fertilization stage of thalli of G. imbricata. As opposite, methionine and MgSO4 did not affect the transcript number of carbohydrate sulfotransferase and galactose-6-sulfurylase. Nonetheless, differential expression was obtained for sulfurylases according to the development stages of thalli of G. imbricata.

Neonatal classic galactosemia-diagnosis, clinical profile and molecular characteristics in unscreened Turkish population.

BACKGROUND: Classic galactosemia (CG) is a rare hereditary disease that can cause serious morbidity and death if it is not diagnosed and treated in early periods of life. Clinical findings usually occur in the neonatal period after the neonate is fed with milk that contains galactose. Most patients are presented with jaundice, hepatomegaly, hypoglycemia and cataracts. OBJECTIVE: We aimed to document the clinical, molecular characteristics, regional estimated incidence and time of diagnosis in newborn with CG. MATERIALS AND METHOD: The data of 63 newborn with CG who were diagnosed and followed up between January 2011 and January 2018 were analyzed retrospectively. RESULTS: During the study period, 63 (33 boys and 30 girls) newborns were diagnosed with CG. The median gestational age was 39 weeks (33-42). Major presenting symptoms were jaundice 90.5% and cataract 41.2%. The mean age at first symptom was 12 ± 7.4 days while the mean age at diagnosis was 18.9 ± 10.6 days. Nearly half of the patients (55.5%) were diagnosed later than the postnatal 15th day. Genetic analysis was performed on 56 patients and homozygous Q188R mutation was found in 92.8%. There were signs of sepsis in 33.3% of the cases. Six patients died due to sepsis. There was consanguinity in 84.1% of the parents and regional estimated incidence was calculated as 1 in 6103 live births. CONCLUSION: Q188R mutation was found in 92.8% of our cases. The regional estimated incidence was found as 1 in 6103 live births. Our study strongly supports that galactosemia should be included in the national newborn screening program.

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24 h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

Simulation of the Interactions of Arginine with Wild-Type GALT Enzyme and the Classic Galactosemia-Related Mutant p.Q188R by a Computational Approach.

Classic galactosemia is an inborn error of metabolism associated with mutations that impair the activity and the stability of galactose-1-phosphate uridylyltransferase (GALT), catalyzing the third step in galactose metabolism. To date, no treatments (including dietary galactose deprivation) are able to prevent or alleviate the long-term complications affecting galactosemic patients. Evidence that arginine is able to improve the activity of the human enzyme expressed in a prokaryotic model of classic galactosemia has induced researchers to suppose that this amino acid could act as a pharmacochaperone, but no effects were detected in four galactosemic patients treated with this amino acid. Given that no molecular characterizations of the possible effects of arginine on GALT have been performed, and given that the samples of patients treated with arginine are extremely limited for drawing definitive conclusions at the clinical level, we performed computational simulations in order to predict the interactions (if any) between this amino acid and the enzyme. Our results do not support the possibility that arginine could function as a pharmacochaperone for GALT, but information obtained by this study could be useful for identifying, in the future, possible pharmacochaperones for this enzyme.

Analysis of the Structure-Function-Dynamics Relationships of GALT Enzyme and of Its Pathogenic Mutant p.Q188R: A Molecular Dynamics Simulation Study in Different Experimental Conditions.

The third step of the catabolism of galactose in mammals is catalyzed by the enzyme galactose-1-phosphate uridylyltransferase (GALT), a homodimeric enzyme with two active sites located in the proximity of the intersubunit interface. Mutations of this enzyme are associated to the rare inborn error of metabolism known as classic galactosemia; in particular, the most common mutation, associated with the most severe phenotype, is the one that replaces Gln188 in the active site of the enzyme with Arg (p.Gln188Arg). In the past, and more recently, the structural effects of this mutation were deduced on the static structure of the wild-type human enzyme; however, we feel that a dynamic view of the proteins is necessary to deeply understand their behavior and obtain tips for possible therapeutic interventions. Thus, we performed molecular dynamics simulations of both wild-type and p.Gln188Arg GALT proteins in the absence or in the presence of the substrates in different conditions of temperature. Our results suggest the importance of the intersubunit interactions for a correct activity of this enzyme and can be used as a starting point for the search of drugs able to rescue the activity of this enzyme in galactosemic patients.

Unique active site formation in a novel galactose 1-phosphate uridylyltransferase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

A gene encoding galactose 1-phosphate uridylyltransferase (GalT) was identified in the hyperthermophilic archaeon Pyrobaculum aerophilum. The gene was overexpressed in Escherichia coli, after which its product was purified and characterized. The expressed enzyme was highly thermostable and retained about 90% of its activity after incubation for 10 minutes at temperatures up to 90°C. Two different crystal structures of P. aerophilum GalT were determined: the substrate-free enzyme at 2.33 Å and the UDP-bound H140F mutant enzyme at 1.78 Å. The main-chain coordinates of the P. aerophilum GalT monomer were similar to those in the structures of the E. coli and human GalTs, as was the dimeric arrangement. However, there was a striking topological difference between P. aerophilum GalT and the other two enzymes. In the E. coli and human enzymes, the N-terminal chain extends from one subunit into the other and forms part of the substrate-binding pocket in the neighboring subunit. By contrast, the N-terminal chain in P. aerophilum GalT extends to the substrate-binding site in the same subunit. Amino acid sequence alignment showed that a shorter surface loop in the N-terminal region contributes to the unique topology of P. aerophilum GalT. Structural comparison of the substrate-free enzyme with UDP-bound H140F suggests that binding of the glucose moiety of the substrate, but not the UDP moiety, gives rise to a large structural change around the active site. This may in turn provide an appropriate environment for the enzyme reaction.

Discovery of Novel Inhibitors Targeting Multi-UDP-hexose Pyrophosphorylases as Anticancer Agents.

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC50 of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC50 of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.

Two Lithuanian Cases of Classical Galactosemia with a Literature Review: A Novel GALT Gene Mutation Identified.

Galactosemia is a rare autosomal recessive genetic disorder that causes impaired metabolism of the carbohydrate galactose. This leads to severe liver and kidney insufficiency, central nervous system damage and long-term complications in newborns. We present two clinical cases of classical galactosemia diagnosed at the Lithuanian University of Health Sciences (LUHS) Kaunas Clinics hospital and we compare these cases in terms of clinical symptoms and genetic variation in the GALT gene. The main clinical symptoms were jaundice and hepatomegaly, significant weight loss, and lethargy. The clinical presentation of the disease in Patient 1 was more severe than that in Patient 2 due to liver failure and E. coli-induced sepsis. A novel, likely pathogenic GALT variant NM_000155.4:c.305T>C (p.Leu102Pro) was identified and we believe it could be responsible for a more severe course of the disease, although further study is needed to confirm this. It is very important to suspect and diagnose galactosemia as early in its course as possible, and introduce lactose-free formula into the patient's diet. Wide-scale newborn screening and genetic testing are particularly crucial for the early detection of the disease.

Effect of genotype on galactose-1-phosphate in classic galactosemia patients.

Impaired activity of galactose-1-phosphate uridyltransferase (GALT) causes classic galactosemia (OMIM 230400), characterized by the accumulation of galactose-1-phosphate (GAL1P) in patients' red blood cells (RBCs). Our recent study demonstrated a correlation between RBC GAL1P and long-term outcomes in galactosemia patients. Here, we analyze biochemical and molecular results in 77 classic galactosemia patients to evaluate the association between GALT genotypes and GAL1P concentration in RBCs. Experimental data from model organisms were also included to assess the correlation between GAL1P and predicted residual activity of each genotype. Although all individuals in this study showed markedly reduced RBC GALT activity, we observed significant differences in RBC GAL1P concentrations among galactosemia genotypes. While levels of GAL1P on treatment did not correlate with RBC GALT activities (p = 0.166), there was a negative nonlinear correlation between mean GAL1P concentrations and predicted residual enzyme activity of genotype (p = 0.004). These studies suggest that GAL1P levels in RBCs on treatment likely reflect the overall functional impairment of GALT in patients with galactosemia.

The galactose-induced decrease in phosphate levels leads to toxicity in yeast models of galactosemia.

Classic galactosemia is an inborn error of metabolism caused by deleterious mutations in the GALT gene. A number of evidences indicate that the galactose-1-phosphate accumulation observed in patient cells is a cause of toxicity in this disease. Nevertheless, the consequent molecular events caused by the galactose-1-phosphate accumulation remain elusive. Here we show that intracellular inorganic phosphate levels decreased when yeast models of classic galactosemia were exposed to galactose. The decrease in phosphate levels is probably due to the trapping of phosphate in the accumulated galactose-1-phosphate since the deletion of the galactokinase encoding gene GAL1 suppressed this phenotype. Galactose-induced phosphate depletion caused an increase in glycogen content, an expected result since glycogen breakdown by the enzyme glycogen phosphorylase is dependent on inorganic phosphate. Accordingly, an increase in intracellular phosphate levels suppressed the galactose effect on glycogen content and conferred galactose tolerance to yeast models of galactosemia. These results support the hypothesis that the galactose-induced decrease in phosphate levels leads to toxicity in galactosemia and opens new possibilities for the development of better treatments for this disease.

The UDP-glucose pyrophosphorylase from Giardia lamblia is redox regulated and exhibits promiscuity to use galactose-1-phosphate.

BACKGROUND: Giardia lamblia is a pathogen of humans and other vertebrates. The synthesis of glycogen and of structural oligo and polysaccharides critically determine the parasite's capacity for survival and pathogenicity. These characteristics establish that UDP-glucose is a relevant metabolite, as it is a main substrate to initiate varied carbohydrate metabolic routes. RESULTS: Herein, we report the molecular cloning of the gene encoding UDP-glucose pyrophosphorylase from genomic DNA of G. lamblia, followed by its heterologous expression in Escherichia coli. The purified recombinant enzyme was characterized to have a monomeric structure. Glucose-1-phosphate and UTP were preferred substrates, but the enzyme also used galactose-1-phosphate and TTP. The catalytic efficiency to synthesize UDP-galactose was significant. Oxidation by physiological compounds (hydrogen peroxide and nitric oxide) inactivated the enzyme and the process was reverted after reduction by cysteine and thioredoxin. UDP-N-acetyl-glucosamine pyrophosphorylase, the other UTP-related enzyme in the parasite, neither used galactose-1-phosphate nor was affected by redox modification. CONCLUSIONS: Our results suggest that in G. lamblia the UDP-glucose pyrophosphorylase is regulated by oxido-reduction mechanism. The enzyme exhibits the ability to synthesize UDP-glucose and UDP-galactose and it plays a key role providing substrates to glycosyl transferases that produce oligo and polysaccharides. GENERAL SIGNIFICANCE: The characterization of the G. lamblia UDP-glucose pyrophosphorylase reinforces the view that in protozoa this enzyme is regulated by a redox mechanism. As well, we propose a new pathway for UDP-galactose production mediated by the promiscuous UDP-glucose pyrophosphorylase of this organism.

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.

Gestational dating by metabolic profile at birth: a California cohort study.

BACKGROUND: Accurate gestational dating is a critical component of obstetric and newborn care. In the absence of early ultrasound, many clinicians rely on less accurate measures, such as last menstrual period or symphysis-fundal height during pregnancy, or Dubowitz scoring or the Ballard (or New Ballard) method at birth. These measures often underestimate or overestimate gestational age and can lead to misclassification of babies as born preterm, which has both short- and long-term clinical care and public health implications. OBJECTIVE: We sought to evaluate whether metabolic markers in newborns measured as part of routine screening for treatable inborn errors of metabolism can be used to develop a population-level metabolic gestational dating algorithm that is robust despite intrauterine growth restriction and can be used when fetal ultrasound dating is not available. We focused specifically on the ability of these markers to differentiate preterm births (PTBs) (<37 weeks) from term births and to assign a specific gestational age in the PTB group. STUDY DESIGN: We evaluated a cohort of 729,503 singleton newborns with a California birth in 2005 through 2011 who had routine newborn metabolic screening and fetal ultrasound dating at 11-20 weeks' gestation. Using training and testing subsets (divided in a ratio of 3:1) we evaluated the association among PTB, target newborn characteristics, acylcarnitines, amino acids, thyroid-stimulating hormone, 17-hydroxyprogesterone, and galactose-1-phosphate-uridyl-transferase. We used multivariate backward stepwise regression to test for associations and linear discriminate analyses to create a linear function for PTB and to assign a specific week of gestation. We used sensitivity, specificity, and positive predictive value to evaluate the performance of linear functions. RESULTS: Along with birthweight and infant age at test, we included 35 of the 51 metabolic markers measured in the final multivariate model comparing PTBs and term births. Using a linear discriminate analyses-derived linear function, we were able to sort PTBs and term births accurately with sensitivities and specificities of ≥95% in both the training and testing subsets. Assignment of a specific week of gestation in those identified as PTBs resulted in the correct assignment of week ±2 weeks in 89.8% of all newborns in the training and 91.7% of those in the testing subset. When PTB rates were modeled using the metabolic dating algorithm compared to fetal ultrasound, PTB rates were 7.15% vs 6.11% in the training subset and 7.31% vs 6.25% in the testing subset. CONCLUSION: When considered in combination with birthweight and hours of age at test, metabolic profile evaluated within 8 days of birth appears to be a useful measure of PTB and, among those born preterm, of specific week of gestation ±2 weeks. Dating by metabolic profile may be useful in instances where there is no fetal ultrasound due to lack of availability or late entry into care.

How strict is galactose restriction in adults with galactosaemia? International practice.

Dietary management of 418 adult patients with galactosaemia (from 39 centres/12 countries) was compared. All centres advised lactose restriction, 6 restricted galactose from galactosides ± fruits and vegetables and 12 offal. 38% (n=15) relaxed diet by: 1) allowing traces of lactose in manufactured foods (n=13) or 2) giving fruits, vegetables and galactosides (n=2). Only 15% (n=6) calculated dietary galactose. 32% of patients were lost to dietetic follow-up. In adult galactosaemia, there is limited diet relaxation.

GALT protein database: querying structural and functional features of GALT enzyme.

Knowledge of the impact of variations on protein structure can enhance the comprehension of the mechanisms of genetic diseases related to that protein. Here, we present a new version of GALT Protein Database, a Web-accessible data repository for the storage and interrogation of structural effects of variations of the enzyme galactose-1-phosphate uridylyltransferase (GALT), the impairment of which leads to classic Galactosemia, a rare genetic disease. This new version of this database now contains the models of 201 missense variants of GALT enzyme, including heterozygous variants, and it allows users not only to retrieve information about the missense variations affecting this protein, but also to investigate their impact on substrate binding, intersubunit interactions, stability, and other structural features. In addition, it allows the interactive visualization of the models of variants collected into the database. We have developed additional tools to improve the use of the database by nonspecialized users. This Web-accessible database (http://bioinformatica.isa.cnr.it/GALT/GALT2.0) represents a model of tools potentially suitable for application to other proteins that are involved in human pathologies and that are subjected to genetic variations.