Methimazole, thyroid hormone replacement, and lipogenic enzyme gene expression in broilers

The purpose of this experiment was to determine the possible relationship between certain indices of lipid metabolism and specific gene expression in chickens fed methimazole to simulate hypothyroidism. Male broiler chickens (Gallus gallus) growing from 7 to 28 days of age were fed diets containing 18% crude protein and either 0 or 1 g methimazole per kilogram of diet. At 28 days, these two groups were further subdivided into groups receiving 18% crude protein diets containing either 0 or 1 mg triiodothyronine (T3) per kilogram. Birds were sampled at 28, 30, and 33 days. Measurements taken included in vitro lipogenesis (IVL), malic enzyme (ME) activity, isocitrate dehydrogenase, aspartate amino transferase, and the expression of the genes for ME, fatty acid synthase (FAS), and acetyl coenzyme carboxylase (ACC). Hypothyroidism decreased IVL and ME at 28 days of age; however, T3 supplementation for 2 days restored both IVL and ME. Paradoxically, continuing T3 replenishment for an additional 3 days decreased IVL but did not decrease ME activity. In contrast, supplemental T3 decreased IVL in euthyroid birds, regardless of the dosing interval, but had no effect on ME activity. Although methimazole decreased ME gene expression, there was only a transitory relationship between enzyme activity and gene expression when plasma T3 was restored with exogenous T3. These data may help to explain some of the apparent reported dichotomies in lipid metabolism elicited by changes in the thyroid state of animals. In addition, most metabolic changes in response to feeding T3 occurred within 2 to 5 days, suggesting that changes in intermediary metabolism preceded morphological changes. In conclusion, the thyroid state of the animal will determine responses to exogenous T3.     

The pathogenesis of transaldolase deficiency

The signaling networks that mediate cell growth, differentiation, and survival are dependent on complex metabolic and redox pathways. Metabolism of glucose through the pentose phosphate pathway (PPP) fulfills two unique functions: formation of ribose 5-phosphate for the synthesis of nucleotides, RNA, and DNA in support cell growth and formation of NADPH for biosynthetic reactions and neutralization of reactive oxygen intermediates (ROI). Balancing of NADPH and ROI levels by the PPP enzyme transaldolase (TAL) regulates the mitochondrial trans-membrane potential (Deltapsi(m)), a critical checkpoint of ATP synthesis and cell survival. While complete deficiency of glucose 6-phosphate dehydrogenase (G6PD) or transketolase (TK) is lethal, TAL-deficient mice developed normally with the exception of male sterility due to structural and functional damage of sperm cell mitochondria. Recently, two cases of complete TAL deficiency have been reported in patients with liver cirrhosis which results from increased cell death of hepatocytes. Delineation of the cell type-specific role that TAL plays in the PPP and cell death signal processing will be critical for understanding the pathogenesis of TAL deficiency.     

V490M, a common mutation in 3-phosphoglycerate dehydrogenase deficiency, causes enzyme deficiency by decreasing the yield of mature enzyme.

A deficiency of 3-phosphoglycerate dehydrogenase (PHGDH) is a disorder of serine biosynthesis identified in children with congenital microcephaly, seizures, and severe psychomotor retardation. We report here the identification of the 1468G-->A (V490M) mutation of this gene in two siblings of an Ashkenazi Jewish family, providing further evidence that the V490M mutation is a common, panethnic cause of this deficiency. Using a novel, DNA-based diagnostic test, the mutation was not detected in 400 non-Jewish controls; one heterozygote was found among 400 persons of Ashkenazi Jewish ethnicity. Extensive biochemical studies were undertaken to characterize the effect of this mutation on enzyme activity, turnover, and stability. The V490M PHGDH yielded less than 35% of the activity observed for the wild-type enzyme when overexpressed by transient transfection or when comparing the endogenous activity in fibroblast cells from the patients with controls. Immunoblotting studies showed a comparable reduction in the level of immunoreactive PHGDH in cells expressing the mutant enzyme. Pulse-chase experiments with metabolically labeled PHGDH indicated that this resulted from an increased rate of degradation of the mutant enzyme following its synthesis. Thermolability analyses of mutant and wild-type enzyme activity revealed no significant differences. While others have proposed that the V490M mutation decreases the V(max) of the enzyme, we conclude that this mutation impairs the folding and/or assembly of PHGDH but has minimal effects on the activity or stability of that portion of the V490M mutant that reaches a mature conformation.     

The role of mannosylated enzyme and the mannose receptor in enzyme replacement therapy

Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. LAL defects cause Wolman disease (WD) and CE storage disease (CESD). An LAL null (lal-/-) mouse model closely mimics human WD/CESD, with hepatocellular, Kupffer cell and other macrophage, and adrenal cortical storage of CEs and TGs. The effect on the cellular targeting of high-mannose and complex oligosaccharide-type oligosaccharide chains was tested with human LAL expressed in Pichia pastoris (phLAL) and CHO cells (chLAL), respectively. Only chLAL was internalized by cultured fibroblasts, whereas both chLAL and phLAL were taken up by macrophage mannose receptor (MMR)-positive J774E cells. After intraperitoneal injection into lal-/- mice, phLAL and chLAL distributed to macrophages and macrophage-derived cells of various organs. chLAL was also detected in hepatocytes. Ten injections of either enzyme over 30 d into 2- and 2.5-mo-old lal-/- mice produced normalization of hepatic color, decreased liver weight (50%-58%), and diminished hepatic cholesterol and TG storage. Lipid accumulations in macrophages were diminished with either enzyme. Only chLAL cleared lipids in hepatocytes. Mice double homozygous for the LAL and MMR deficiences (lal-/-;MMR-/-) showed phLAL uptake into Kupffer cells and hepatocytes, reversal of macrophage histopathology and lipid storage in all tissues, and clearance of hepatocytes. These results implicate MMR-independent and mannose 6-phosphate receptor-independent pathways in phLAL uptake and delivery to lysosomes in vivo. In addition, these studies show specific cellular targeting and physiologic effects of differentially oligosaccharide-modified human LALs mediated by MMR and that lysosomal targeting of mannose-terminated glycoproteins occurs and storage can be eliminated effectively without MMR.     

Cognitive function in growth hormone deficiency and growth hormone replacement

There is converging evidence from neuropsychological studies that growth hormone (GH) is associated with cognitive function. The aim of the current study was to review the existing neuropsychological literature for studies in which cognitive assessment had been conducted in patients with GH deficiency (GHD), and where change in cognitive function had been assessed following treatment with GH. Studies that have investigated relationships between GH and cognitive function and those that have developed methodological and statistical approaches that could be useful in future GH studies were identified. In this review, GH levels were found to be associated with cognitive function. Untreated individuals with GHD showed reliable impairment in memory and attentional functions when compared with matched controls. Appropriately designed prospective studies also indicated that cognitive function improved with GH treatment. It was concluded that individuals with GHD do show cognitive impairment and that this is ameliorated to some extent by GH treatment. It is now important to establish the clinical importance of these findings, and further work is required to understand better the nature, magnitude and meaning of GH-related cognitive impairments and improvements.     

Genotype, enzyme activity, glutathione level, and clinical phenotype in patients with glutathione synthetase deficiency

Glutathione synthetase (GS) deficiency is a rare autosomal recessive disorder. The clinical phenotype varies widely, and nearly 30 different mutations in the GSS gene have been identified. In the present study, genotype, enzyme activity, metabolite levels and clinical phenotype were evaluated in 41 patients from 33 families. From some of the patients, data on glutathione (GSH) levels and -glutamylcysteine levels in cultured fibroblasts were also available. Twenty-seven different mutations were found: 14 missense, 9 splice, 2 deletions, 1 insertion and 1 nonsense mutation. Twenty-three patients were homozygous and 18 were compound heterozygous. The moderate and severe clinical phenotypes could not be distinguished based on enzyme activity, GSH or -glutamylcysteine levels in cultured fibroblasts. However, in fibroblasts, the residual GS activity was correlated with the GSH level. All mutations causing frameshifts, premature stop codons or aberrant splicing were associated with moderate or severe clinical phenotypes including haemolytic anaemia, 5-oxoprolinuria, and (in several forms) neurodevelopmental signs. The data indicate that additional genetic or environmental factors modify at least the moderate and severe phenotypes and that the clinical classification given to the patients may be influenced by variation in follow-up. The type of mutation involved can, to some extent, predict a mild versus a more severe phenotype.Electronic Supplementary Material Suplementary material is available for this article at .Electronic database information: Online Mendelian Inheritance in Man, (MIM 266130 for GS deficiency); GenBank, ( nos. AL133324.13 for genomic DNA and NM_000178.2 for mRNA were used for sequence referencing)     

Production in yeast of alpha-galactosidase A,a lysosomal enzyme applicable to enzyme replacement therapy for Fabry disease

A mammalian-like sugar moiety was created in glycoprotein by Saccharomyces cerevisiae in combination with bacterial alpha-mannosidase to produce a more economic enzyme replacement therapy for patients with Fabry disease. We introduced the human alpha-galactosidase A (alpha-GalA) gene into an S. cerevisiae mutant that was deficient in the outer chains of N-linked mannan. The recombinant alpha-GalA contained both neutral (Man(8)GlcNAc(2)) and acidic ([Man-P](1-2)Man(8)GlcNAc(2)) sugar chains. Because an efficient incorporation of alpha-GalA into lysosomes of human cells requires mannose-6-phosphate (Man-6-P) residues that should be recognized by the specific receptor, we trimmed down the sugar chains of the alpha-GalA by a newly isolated bacterial alpha-mannosidase. Treatment of the alpha-GalA with the alpha-mannosidase resulted in the exposure of a Man-6-P residue on a nonreduced end of oligosaccharide chains after the removal of phosphodiester-linked nonreduced-end mannose. The treated alpha-GalA was efficiently incorporated into fibroblasts derived from patients with Fabry disease. The uptake was three to four times higher than that of the nontreated alpha-GalA and was inhibited by the addition of 5 mM Man-6-P. Incorporated alpha-GalA was targeted to the lysosome, and hydrolyzed ceramide trihexoside accumulated in the Fabry fibroblasts after 5 days. This method provides an effective and economic therapy for many lysosomal disorders, including Fabry disease.     

Immunoreactive enzyme protein in medium-chain acyl-CoA dehydrogenase deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of mitochondrial fatty acid oxidation. To determine if immunoreactive enzyme protein is present in patients with MCAD deficiency, we studied cultured skin fibroblasts from patients with the 985 point mutation, present in about 85% of cases, and cell lines from patients in which the point mutation is not present or only involves one allele. Immunoblotting studies, using a polyclonal antibody to the purified protein, showed an absence of immunoreactive protein in mitochondrial fractions prepared from fibroblasts from MCAD-deficient patients. To determine whether MCAD protein accumulated in the cytosol because of impaired transport into the mitochondria, we immunoprecipitated MCAD protein from the fibroblast homogenate. MCAD protein was detected in the immunoprecipitates from controls, but not in those from the MCAD-deficient patients. These results suggest that either the MCAD protein is not synthesised or, if produced, it is rapidly degraded.     

The crystal structure and mode of action of trans-sialidase,a key enzyme in Trypanosoma cruzi pathogenesis

Trans-sialidases (TS) are GPI-anchored surface enzymes expressed in specific developmental stages of trypanosome parasites like Trypanosoma cruzi, the etiologic agent of Chagas disease, and T. brucei, the causative agent of sleeping sickness. TS catalyzes the transfer of sialic acid residues from host to parasite glycoconjugates through a transglycosidase reaction that appears to be critical for T. cruzi survival and cell invasion capability. We report here the structure of the T. cruzi trans-sialidase, alone and in complex with sugar ligands. Sialic acid binding is shown to trigger a conformational switch that modulates the affinity for the acceptor substrate and concomitantly creates the conditions for efficient transglycosylation. The structure provides a framework for the structure-based design of novel inhibitors with potential therapeutic applications.     

G-6-PD Poznań, variant with severe enzyme deficiency.

A new variant of G-6-PD with severe enzyme dificiency without chronic hemolysis is characterized. The biochemical properties of the variant closely resemble those of the G-6-PD Ramat-Gan described in a case of chronic non-spherocytic hemolytic anemia. As the family data on linking of chronic hemolysis with Ramat-Gan variant are lacking, the differentiation of the variants on the basis of clinical manifestations is not well-founded.     

The different extent of B and T cell immune reconstitution after hematopoietic stem cell transplantation and enzyme replacement therapies in SCID patients with adenosine deaminase deficiency

The lack of adenosine deaminase (ADA) leads to the accumulation of toxic metabolites, resulting in SCID. If the disease is left untreated, it is likely to have a fatal outcome in early infancy. Because hematopoietic stem cell transplantation (HSCT) and enzyme replacement therapy with pegylated bovine ADA (PEG-ADA) are both provided in our hospital, we undertook a retrospective longitudinal comparative study of the extent of lymphocyte recovery in two groups of treated ADA-SCID children. Together with classical immunological parameters, we quantified the output of the new B and T cells from the production sites using the κ-deleting recombination excision circle and TCR excision circle assay, and we monitored T cell repertoire diversification. We found that immune reconstitution was different following the two treatments. The stable production of κ-deleting recombination excision circle(+) lymphocytes sustained an increase in B cell number in HSCT-treated patients, whereas in PEG-ADA-treated patients, it was accompanied by a significant and progressive decrease in circulating CD19(+) lymphocytes, which never reached the levels observed in age-matched children. The mobilization of TCR excision circle(+) cells, though lower than in controls, was stable with time after HSCT treatment, leading to a constant peripheral T cell number and to the diversification of the T cell repertoire; however, it was compromised in children receiving prolonged PEG-ADA therapy, whose T cells showed progressively narrowing T cell repertoires.     

Lysosomal leukocyte beta-D-glucuronidase during enzyme replacement therapy in Fabry disease

OBJECTIVE: Fabry disease results from a deficiency in the activity of alpha-d-galactosidase A and subsequent accumulation of neutral glycosphingolipids in lysosomes. This study investigated whether lysosomal enzymes can indicate biochemical changes in the lysosomal apparatus induced by enzyme replacement therapy (ERT). DESIGN AND METHODS: Eight patients were monitored by clinical and biochemical tests and several lysosomal glycohydrolases were measured in plasma and leucocytes. RESULTS: Before starting ERT, beta-d-glucuronidase in leukocytes was markedly increased. After 1 month of therapy, enzyme levels dropped in all patients. In the patients who regularly followed the therapy, the enzyme levels remained stable for the next 20 months. In one patient who interrupted therapy for 2 months, the enzyme levels rose again. CONCLUSIONS: Lysosomal enzymes can be useful for monitoring biochemical changes in patients with Fabry disease receiving ERT. Though these findings refer to only a small number of patients, the correlation between beta-d-glucuronidase levels and ERT is interesting and might serve as a basis for further studies to define the potential of this enzyme in monitoring the effects of ERT in lysosomal storage disorders.     

Possible hyperaldosteronism and discrepancy in enzyme activity deficiency in adrenal and gonadal glands in Japanese patients with 17 alpha-hydroxylase deficiency

We reviewed the pathophysiology of our previously reported female patient who had glucocorticoid-responsive hyperaldosteronism and was treated successfully with daily dose of dexamethasone (Dex) for 21 years. In this present study, the possibility that the patient may have 17 alpha-hydroxylase deficiency (17-OH-D) mainly in the adrenal could not be ruled out. We therefore reviewed 31 Japanese patients diagnosed as having 17-OH-D with suppressed plasma renin activity reported in Japan. Among these patients, 9 were found to have a high plasma aldosterone (Ald) concentration (PAC) (group I). Twenty-one patients had either normal or low-normal PAC and the remaining patient had low urine Ald (group II). The slight cross-reactivity of the anti-Ald-antibodies used with 17-deoxy-steroids such as progesterone, 11-deoxycorticosterone and corticosterone which were increased in both groups did not explain the increased PAC in group I. In the patients in group I and group II with high-normal basal PAC, PAC further increased after ACTH and was suppressed by Dex. PAC in 2 group I patients, however, did not respond to angiotensin-II or angiotensin-III infusion. PAC in patients in group II with low or low-normal basal PAC responded equivocally to ACTH and Dex. The basal plasma cortisol in group I was lower than in group II, and plasma cortisol level after ACTH in group I appeared to remain at a lower level than that in group II patients. Among the study subjects, 28 showed a negative correlation between basal PAC and plasma cortisol. A possible discrepancy in the deficiency of 17 alpha-hydroxylase activity in adrenal and gonadal glands was also suggested in three 17-OH-D patients. The pathophysiology of Ald secretion and discrepancy in the deficiency of the enzyme activities in both glands in 17-OH-D patients was discussed.     

Amidohydrolases of the reductive pyrimidine catabolic pathway purification, characterization, structure, reaction mechanisms and enzyme deficiency

In the reductive pyrimidine catabolic pathway uracil and thymine are converted to beta-alanine and beta-aminoisobutyrate. The amidohydrolases of this pathway are responsible for both the ring opening of dihydrouracil and dihydrothymine (dihydropyrimidine amidohydrolase) and the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate (beta-alanine synthase). The review summarizes what is known about the properties, kinetic parameters, three-dimensional structures and reaction mechanisms of these proteins. The two amidohydrolases of the reductive pyrimidine catabolic pathway have unrelated folds, with dihydropyrimidine amidohydrolase belonging to the amidohydrolase superfamily while the beta-alanine synthase from higher eukaryotes belongs to the nitrilase superfamily. beta-Alanine synthase from Saccharomyces kluyveri is an exception to the rule and belongs to the Acyl/M20 family.     

Phenylalanine ammonia-lyase immobilized in semipermeable microcapsules for enzyme replacement in phenylketonuria

Phenylalanine ammonia-lyase immobilized within semipermeable microcapsules has an assayed enzyme activity which is 20% +/- 4% of the enzyme in free solution. The Km for the immobilized enzyme remained the same as that of the free enzyme. The pH optimum also remained unchanged at pH 8.5 +/- 1.0. At the lower pH range, enzyme activity is higher for the immobilized enzyme. Daily oral administration of microencapsulated phenylalanine ammonia-lyase to phenylketonuric rats decreased the systemic phenylalanine level by 35 +/- 8% in 2 days (P less than 0.05) and by 75 +/- 8% in 7 days (P less than 0.001).