Static and dynamic interactions between GALK enzyme and known inhibitors: guidelines to design new drugs for galactosemic patients.

The search for inhibitors of galactokinase (GALK) enzyme is interesting for their possible therapeutic application capable to alleviate symptoms in people with classic galactosemia. Several high-throughput screenings in the past have found candidate ligands showing a moderate affinity for GALK. Computational analysis of the binding mode of these compounds in comparison to their target protein has been performed only on crystallographic static structures, therefore missing the evolution of the complex during time. In this work, we applied static and dynamics simulations to analyze the interactions between GALK and its potential inhibitors, while taking into account the temporal evolution of the complexes. The collected data allowed us to identify the most important and persistent anchoring points of the known active site and of the newly identified secondary cavity. These data will be of use to increase the specificity and the affinity of a new generation of GALK inhibitors.

Nutrigenomics in postgenomic era.

The last years modified major our conceptions about nutrition. These revolutionary changes were produced by implementation of new techniques of functional genomics. The nutrigenomics and nutrigenomics provide powerful approaches to unravel the complex relationships between bioactive molecules, genetic polymorphisms and biological system and can give rise to personalized nutrition and dietary recommendations. In monogenic diseases (phenylketonuria, galactosemia, lactose intolerance etc.) diet influence phenotypic expression and nutrigenomics will improve the prevention or treatment by the early identification of specific mutations or haplotype combinations that modulate dietary response in affected subjects. In the multifactor diseases, like cardiovascular diseases (CVD), obesity, type II diabetes mellitus or cancer, the nutrigenomics approach has begun to reveal that some of them are susceptible to dietary intervention and may modulate the onset and progression of disorders.

Inhibition of caspase-1/interleukin-1beta signaling prevents degeneration of retinal capillaries in diabetes and galactosemia.

The proinflammatory cytokine, interleukin (IL)-1beta, is known to induce vascular dysfunction and cell death. We investigated the role of IL-1beta and caspase-1 (the enzyme that produces it) in diabetes-induced degeneration of retinal capillaries. Caspase-1 activity is increased in retinas of diabetic and galactosemic mice and diabetic patients. First, we investigated the effect of agents known to inhibit caspase-1 (minocycline and tetracycline) on IL-1beta production and retinal capillary degeneration in diabetic and galactose-fed mice. Second, we examined the effect of genetic deletion of the IL-1beta receptor on diabetes-induced caspase activities and retinal capillary degeneration. Diabetic and galactose-fed mice were injected intraperitoneally with minocycline or tetracycline (5 mg/kg). At 2 months of diabetes, minocycline inhibited hyperglycemia-induced caspase-1 activity and IL-1beta production in the retina. Long-term administration of minocycline prevented retinal capillary degeneration in diabetic (6 months) and galactose-fed (13 months) mice. Tetracycline inhibited hyperglycemia-induced caspase-1 activity in vitro but not in vivo. Mice deficient in the IL-1beta receptor were protected from diabetes-induced caspase activation and retinal pathology at 7 months of diabetes. These results indicate that the caspase-1/IL-1beta signaling pathway plays an important role in diabetes-induced retinal pathology, and its inhibition might represent a new strategy to inhibit capillary degeneration in diabetic retinopathy.

Pyruvate inhibits galactosemic changes in cultured cat lens epithelial cells.

An attempt was made to maintain cat lens epithelial cells (CLEC) in culture and study the morphology, growth and survival of these cells in vitro. The influence of incorporation of galactose (30 mM) into the culture medium on the morphology and biochemistry of CLEC in the primary culture was then investigated. To establish the effect of galactose on CLEC, various biochemical parameters associated with galactosemic cataract such as aldose reductase (AR), Na+K+ATPase, glutathione, polyol and soluble/insoluble proteins were estimated after 24 h of incubation. The effect of pyruvate (5 mM), a 'physiological antioxidant', on the changes induced by galactose in CLEC was studied. CLEC in culture showed regular hexagonal cells with prominent nuclei. The CLEC culture attained confluency in 11 days during primary culture and semiconfluency in 14 days in two subsequent passages. Vacuolization and significantly raised AR activity, polyol levels and insoluble protein contents were observed; they had no effect on Na+K+ATPase and soluble protein after 24 h of incubation in the culture medium with galactose. Supplementation of pyruvate (5 mM) resulted in a lesser number of vacuoles together with a positive modulation of these parameters.

Two novel glucose-6-phosphate dehydrogenase variants found in newborn mass-screening for galactosaemia.

UNLABELLED: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive disorder in which haemolytic anaemia is the major symptom. The Beutler spot test employed in mass-screening for galactosaemia in newborns requires several intrinsic erythrocyte enzymes such as G6PD for its reaction and can theoretically detect G6PD deficiency apart from galactose-1-phosphate uridyltransferase deficiency. In this study, we detected two patients with G6PD deficiency using the quantitative Beutler test which was recently developed in our laboratory. Both patients lacked erythrocyte G6PD activity but exhibited no clinical symptoms. Molecular analysis in patients 1 and 2 revealed two novel missense mutations of C853T causing R285C and A1220C causing K407T, respectively. Molecular rather than enzymatic analysis was required in familial studies to detect and diagnose the carrier state. To date these patients have avoided oxidant stress and haemolytic diatheses have not been induced. CONCLUSION: Our results indicate that the quantitative Beutler test can detect glucose-6-phosphate dehydrogenase deficiency of class 1 and 2 and is therefore useful for early intervention and prevention of haemolytic diathesis in patients with this disorder.

Classical galactosaemia in Chinese: A case report and review of disease incidence.

We report a case of galactose-1-phosphate uridyl transferase (GALT) deficiency in a full-term Chinese neonate, who presented with atypical biochemical features of hyperammonaemia in addition to the classical presenting features of jaundice and lethargy after feeding. Red cell GALT activity was virtually absent in the patient while 50% of normal activity was found in parents and a sibling. Mutation screening excluded both Q188R and N314D as the causative mutation in GALT gene, which suggested a possible genetic segregation among ethnic groups. Data from a Taiwan screening program suggested that the incidence of the disease was approximately 1 in 400 000 in the Chinese population which was a sixth of that in Caucasian populations.

Newborn screening for galactosemia: ultramicro assay for galactose-1-phosphate-uridyltransferase activity.

An enzymatically optimized, miniaturized (20 microl) fluorimetric assay of galactose-1-phosphate-uridyltransferase using dried blood spots for newborn screening is presented. The Beutler reaction principle has been adapted to the microtiter plate technology and acetone/methanol was used for complete deproteinization. A special ultramicro multiwell screening plate resistant to organic solvents has been developed and employed. The assay is simple, sensitive and inexpensive, due to small reagent volumes and the low prices of ultramicro screening plates. The reaction is linear with galactose-1-phosphate-uridyltransferase activity up to 120 min of incubation time. It shows low imprecision and good correlation to a quantitative validation test. For standardization the use of plate means or medians of activity or fluorescence values is proposed. Individual blank measurement prevents false negative assessments.

Screening for galactosaemia in Greece.

Galactosaemia appears to be one of the most appropriate disorders for routine newborn screening as almost normal outcome can be achieved in most of the identified cases. Galactose and galactose-1-phosphate were determined using Guthrie cards in a commercial kit based on a colorimetric microassay. Among 199,642 newborns, nine cases with classic galactosaemia, three with epimerase deficiency, six with compound Duarte2/heterozygotes for galactosaemia and four with compound2 Duarte homozygosity were found. Even though the number found among the screened neonates is small because it is such a rare disease, our results indicate one of the highest frequencies of the disease ever reported.

Newborn screening for certain treatable inborn errors of metabolism in Alexandria.

The study was conducted on two groups of newborn infants: Group A; a random sample of 3000 infants attending different Health offices in Alexandria for BCG vaccination. Their ages ranged from 5-120 days with a mean age of 39.9 days. Group B; included all the infants born to high risk families attending the clinic of Human Genetics Department, Medical Research Institute (9 infants; 7 with family history of PKU and 2 with family history of congenital hypothyroidism). Their ages ranged from 7 to 60 days with a mean age of 18 days. The newborn infants of the two groups were screened for three treatable inborn errors of metabolism, phenylketonuria "PKU", galactosemia and congenital hypothyroidism with the aim of early detection and therapy to prevent mental retardation. In group A; one baby with transient hyperphenylalaninemia (HPA) (0.33%) and one presumptive case of galactosemia (0.33%) were found. Initial positive results were found in eleven infants they had high levels of thyroid stimulating hormone (TSH). On reevaluation of nine infants of them they were all euthyroids. In Group B, four infants were detected among the infants of PKU families. After confirmation of these results breast feeding was stopped at once and the infants started their dietary management and were kept on it with follow up and periodic evaluation of the adequacy of treatment.

Galactosaemia--a controversial disorder. Screening & outcome. Ireland 1972-1992.

We reviewed 20 years (from 1972 to 1992) of screening for galactosaemia in Ireland. We looked at a small group of 32 patients followed up in the same centre since diagnosis. 1.2 million babies have been screened with 55 cases of classical galactosaemia and 7 Duarte Variants being detected. The frequency is thus, 1:23,000 and is increased among itinerants to 1:700. The mean age of diagnosis was 6.9 days with 41/62 cases symptomatic at the time. There were 9 deaths, 8 in the first 10 years, six of whom were itinerants. On follow up of the 32 children, who have attended Temple Street Hospital, 13/32 have no detectable complication. Nineteen show either one or a combination of cataracts, speech problems, tremors, abnormal FSH and or LH, delayed mental development and recurring infections. The screening test is the Bacterial Inhibition Assay with Beutler test, an original blood spot as confirmation in presumptive possible cases. The Beutler test is performed urgently in high risk situations. Five classical galactosaemics gave false negative results, 3 because of poor feeding, and two because of soya milk formulas. Screening prevented deaths as 7/84 siblings of our cases were unexpected infant deaths, all but one predating screening. Unexplained delay in screening undermines its effectiveness. Survival is enhanced by aggressive neonatal care. Symptoms are improved on commencing diet, with cataracts regressing. Complications did not correlate with the day of starting diet. Galactose-1-Phosphate and urinary galactitol levels did not correlate with complications, but slit lamp examination has proved a helpful index of adherence to diet. The mechanism of complications is unclear, and it is essential that current research should be aggressively pursued to explain mental deficiency in cases treated either pre-natally or early in post natal life.

Nutritional therapy for pregnant women with a metabolic disorder.

Nutritional therapy is essential for a normal reproductive outcome in phenylketonuric women. In homocystinuria, fetal outcome is good in women whose disorder is responsive to vitamin B6 therapy and is poor in women whose disorder is unresponsive to therapy. Pregnancy in galactosemia is rare because of the almost universal ovarian dysfunction present in female patients with this disorder. Transplantation of the fertilized ovum is a promising possibility for these women. In women with MSUD, there has been only one case of pregnancy reported to date.

Galactosemia produces ARI-preventable nodal changes similar to those of diabetic neuropathy.

The present study was designed to examine the development of structural changes, characteristic of diabetic neuropathy, in chronic galactosemia and their responsiveness to inhibition of the polyol-pathway. Sprague-Dawley rats weighing 70-90 g were given a 50% galactose diet continued for 4 or 8 months. Half of these animals were simultaneously given the aldose reductase inhibitor (ARI) WAY 121-509. ARI-treatment normalized galactitol and myoinositol levels in the sciatic nerve. At 4 months, sciatic nerve conduction velocity (NCV) in galactosemic rats was reduced by 30% which was prevented in ARI-treated rats. At 8 months galactosemia reduced NCV to 58% of control values, while ARI-treatment for 8 months improved NCV to 71% of control values. ARI-treatment prevented in galactosemic rats nodal structural changes characteristic of diabetic neuropathy, whereas axonal atrophy was not affected by ARI-treatment, which may in part account for the only partial prevention of the NCV slowing at 8 months. Nerve fiber regeneration was increased 4-fold in ARI-treated rats compared with untreated galactosemic rats. These data suggest that chronic galactosemia produces a neuropathy structurally similar to diabetic neuropathy. The lack of an ARI-treatment effect on axonal atrophy suggests that this defect is not polyol related in galactosemia.