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We describe neurotransmitter abnormalities in two patients with drug-resistant epilepsy resulting from deleterious de novo mutations in sodium channel genes. Whole exome sequencing identified a de novo SCN2A splice-site mutation (c.2379 +1G>A, p.G1u717Gly.fs*30) resulting in deletion of exon 14, in a 10-year old male with early onset global developmental delay, intermittent ataxia, autism, hypotonia, epileptic encephalopathy and cerebral/cerebellar atrophy. In the cerebrospinal fluid both homovanillic acid and 5-hydroxyindoleacetic acid were significantly decreased; extensive biochemical and genetic investigations ruled out primary neurotransmitter deficiencies and other known inborn errors of metabolism. In an 8-year old female with an early onset intractable epileptic encephalopathy, developmental regression, and progressive cerebellar atrophy, a previously unreported de novo missense mutation was identified in SCN8A (c.5615G>A; p.Arg1872GIn), affecting a highly conserved residue located in the C-terminal of the Na(v)1.6 protein. Aside from decreased homovanillic acid and 5-hydroxyindoleacetic acid, 5-methyltetrahydrofolate was also found to be low. We hypothesize that these channelopathies cause abnormal synaptic mono-amine metabolite secretion/uptake via impaired vesicular release and imbalance in electrochemical ion gradients, which in turn aggravate the seizures. Treatment with oral 5-hydroxytryptophan, L-Dopa/Carbidopa, and a dopa agonist resulted in mild improvement of seizure control in the male case, most likely via dopamine and serotonin receptor activated signal transduction and modulation of glutamatergic, GABA-ergic and glycinergic neurotransmission. Neurotransmitter analysis in other sodium channelopathy patients will help validate our findings, potentially yielding novel treatment opportunities. (C) 2015 Elsevier Inc. All rights reserved

Objective— High-density lipoproteins (HDL) are considered to protect against atherosclerosis in part by facilitating the removal of cholesterol from peripheral tissues. However, factors regulating lipid efflux are incompletely understood. We previously identified a variant in adenosine triphosphate–binding cassette transporter A8 ( ABCA8 ) in an individual with low HDL cholesterol (HDLc). Here, we investigate the role of ABCA8 in cholesterol efflux and in regulating HDLc levels. Approach and Results— We sequenced ABCA8 in individuals with low and high HDLc and identified, exclusively in low HDLc probands, 3 predicted deleterious heterozygous ABCA8 mutations (p.Pro609Arg [P609R], IVS17-2 A>G and p.Thr741Stop [T741X]). HDLc levels were lower in heterozygous mutation carriers compared with first-degree family controls (0.86±0.34 versus 1.17±0.26 mmol/L; P =0.005). HDLc levels were significantly decreased by 29% ( P =0.01) in Abca8b −/− mice on a high-cholesterol diet compared with wild-type mice, whereas hepatic overexpression of human ABCA8 in mice resulted in significant increases in plasma HDLc and the first steps of macrophage-to-feces reverse cholesterol transport. Overexpression of wild-type but not mutant ABCA8 resulted in a significant increase (1.8-fold; P =0.01) of cholesterol efflux to apolipoprotein AI in vitro. ABCA8 colocalizes and interacts with adenosine triphosphate–binding cassette transporter A1 and further potentiates adenosine triphosphate–binding cassette transporter A1–mediated cholesterol efflux. Conclusions— ABCA8 facilitates cholesterol efflux and modulates HDLc levels in humans and mice.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Primary 5-oxoprolinuria (pyroglutamic aciduria) is caused by a genetic defect in the gamma-glutamyl cycle, affecting either glutathione synthetase or 5-oxoprolinase. While several dozens of patients with glutathione synthetase deficiency have been reported, with hemolytic anemia representing the clinical key feature, 5-oxoprolinase deficiency due to OPLAH mutations is less frequent and so far has not attracted much attention. This has prompted us to investigate the clinical phenotype as well as the underlying genotype in patients from 14 families of various ethnic backgrounds who underwent diagnostic mutation analysis following the detection of 5-oxoprolinuria. In all patients with 5-oxoprolinuria studied, bi-allelic mutations in OPLAH were indicated. An autosomal recessive mode of inheritance for 5-oxoprolinase deficiency is further supported by the identification of a single mutation in all 9/14 parent sample sets investigated (except for the father of one patient whose result suggests homozygosity), and the absence of 5-oxoprolinuria in all tested heterozygotes. It is remarkable, that all 20 mutations identified were novel and private to the respective families. Clinical features were highly variable and in several sib pairs, did not segregate with 5-oxoprolinuria. Although a pathogenic role of 5-oxoprolinase deficiency remains possible, this is not supported by our findings. Additional patient ascertainment and long-term follow-up is needed to establish the benign nature of this inborn error of metabolism. It is important that all symptomatic patients with persistently elevated levels of 5-oxoproline and no obvious explanation are investigated for the genetic etiology. (C) 2016 Elsevier Inc. All rights reserved.

We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic and metabolic investigations for acute liver failure failed to yield a diagnosis. Whole exome sequencing revealed a homozygous 12-bp deletion in PCK1 (MIM 614168) encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK); enzymatic studies subsequently confirmed its pathogenic nature. We propose that PEPCK deficiency should be considered in the young child with unexplained liver failure, especially where there are marked, accumulations of TCA cycle metabolites on urine organic acid analysis and/or an amino acid profile with hyperammonaemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-)physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes. (C) 2016 Elsevier Inc. All rights reserved

The lipidation of apoA-I in liver greatly influences HDL biogenesis and plasma HDL levels by stabilizing the secreted apoA-I. Niacin is the most effective lipid-regulating agent clinically available to raise HDL. This study was undertaken to identify regulatory mechanisms of niacin action in hepatic lipidation of apoA-I, a critical event involved in HDL biogenesis. In cultured human hepatocytes (HepG2), niacin increased: association of apoA-I with phospholipids and cholesterol by 46% and 23% respectively, formation of lipid-poor single apoA-I molecule-containing particles up to ∼ 2.4-fold, and pre β 1 and α migrating HDL particles. Niacin dose-dependently stimulated the cell efflux of phospholipid and cholesterol and increased transcription of ABCA1 gene and ABCA1 protein. Mutated DR4, a binding site for nuclear factor liver X receptor alpha (LXR α ) in the ABCA1 promoter, abolished niacin stimulatory effect. Further, knocking down LXR α or ABCA1 by RNA interference eliminated niacin-stimulated apoA-I lipidation. Niacin treatment did not change apoA-I gene expression. The present data indicate that niacin increases apoA-I lipidation by enhancing lipid efflux through a DR4-dependent transcription of ABCA1 gene in HepG2 cells. A stimulatory role of niacin in early hepatic formation of HDL particles suggests a new mechanism that contributes to niacin action to increase the stability of newly synthesized circulating HDL.

In this study, the metabolic network of ectoine by Halomonas venusta DSM 4743 was established. The key nodes to influence the ectoine fermentation in metabolic flux and the basis during optimal control of fermentation process were investigated. The results showed that G6P, α-KG and OAA nodes were the key factors to influence the synthesis of ectoine. The metabolic flux distributions at the key nodes were significantly improved and ectoine concentration was enhanced in ectoine fermentation by adopting monosodium glutamate as the sole carbon and nitrogen sources, feeding monosodium glutamate and supplying oxygen limitedly. The batch fermentation was carried out in 10 L fermentor , the concentration and yield of ectoine was 8.4 g/L and 0.1 g/g, respectively, which were increased by 2.8 and 2 times, by comparison with batch fermentation using glucose as carbon source.

In pharmacological doses, nicotinic acid (niacin) reduces myocardial infarction, stroke and atherosclerosis. The beneficial effects of niacin on lipoproteins are thought to mediate these effects. We hypothesized that niacin inhibits oxidative stress and redox-sensitive inflammatory genes that play a critical role in early atherogenesis. In cultured human aortic endothelial cells (HAEC), niacin increased nicotinamide adenine dinucleotide phosphate (NAD(P)H) levels by 54% and reduced glutathione (GSH) by 98%. Niacin inhibited: (a) angiotensin II (ANG II)-induced reactive oxygen species (ROS) production by 24-86%, (b) low density lipoprotein (LDL) oxidation by 60%, (c) tumor necrosis factor alpha (TNF-alpha)-induced NF-kappaB activation by 46%, vascular cell adhesion molecule-1 (VCAM-1) by 77-93%, monocyte chemotactic protein-1 (MCP-1) secretion by 34-124%, and (d) in a functional assay TNF-alpha-induced monocyte adhesion to HAEC (41-54%). These findings indicate for the first time that niacin inhibits vascular inflammation by decreasing endothelial ROS production and subsequent LDL oxidation and inflammatory cytokine production, key events involved in atherogenesis. Initial data presented herein support the novel concept that niacin has vascular anti-inflammatory and potentially anti-atherosclerotic properties independent of its effects on lipid regulation.

Niacin is an effective agent for raising HDL, but its cellular target sites are largely unknown. We examined effects of niacin on the surface expression of ATP synthase beta chain, a newly described HDL/apolipoprotein A-I (apoA-I) receptor for HDL endocytosis, in HepG2 cells. A significant amount of immunodetectable beta chain was observed on the surface of HepG2 cells, which was competitively displaced by apoA-I. Niacin treatment reduced the surface expression of beta chain in HepG2 cells by approximately 27%, and decreased (125)I-labeled HDL uptake up to approximately 35%. However, nicotinamide, a niacin metabolite that does not have clinical lipid effects, exhibited weaker inhibition on the beta chain cell surface expression, and failed to show inhibitory action on (125)I-labeled HDL uptake. Furthermore, anti-beta chain antibody significantly reduced (125)I-labeled HDL uptake and abolished the inhibitory effect of niacin. Niacin did not change beta chain mRNA expression. These data suggest that niacin inhibits cell surface expression of the ATP synthase beta chain, leading to reduced hepatic removal of HDL protein, thus implicating a potential cellular target for niacin action to raise HDL.

Niacin has been in use for the past 50 years to treat lipid disorders and atherosclerotic cardiovascular disease. We have recently demonstrated that niacin, by selective inhibition of liver diacylglycerol acyltransferae (DGAT)2, a rate-limiting enzyme in triglyceride synthesis, inhibits triglyceride synthesis thereby decreasing the secretion of apolipoprotein (Apo)B-containing very low-density lipoprotein and low-density lipoprotein particles. We have demonstrated that niacin increases ApoAI–high-density lipoprotein (HDL), mainly through the selective inhibition of hepatic uptake and removal of ApoAI–HDL (but not HDL-cholesterol ester). Emerging data from our laboratory provide evidence for the nonlipid-related effects of niacin on vascular oxidative and inflammatory processes involved in atherosclerosis. Although decreased free fatty acid mobilization from adipose tissue via a newly described niacin receptor (HM74A) is reported as a mechanism by which niacin decreases triglycerides, both physiologically ...

Mutations in ABCA1 uniformly decrease plasma HDL-cholesterol (HDL-C) and reduce cholesterol efflux, yet different mutations in ABCA1 result in different phenotypic effects in heterozygotes. For example, truncation mutations result in significantly lower HDL-C and apoliprotein A-I (apoA-I) levels in heterozygotes compared with nontruncation mutations, suggesting that truncation mutations may negatively affect the wild-type allele. To specifically test this hypothesis, we examined ABCA1 protein expression in response to 9-cis-retinoic acid (9-cis-RA) and 22-R-hydroxycholesterol (22-R-OH-Chol) in a collection of human fibroblasts representing eight different mutations and observed that truncation mutations blunted the response to oxysterol stimulation and dominantly suppressed induction of the remaining full-length allele to 5–10% of wild-type levels. mRNA levels between truncation and nontruncation mutations were comparable, suggesting that ABCA1 expression was suppressed at the protein level. Dominant negative activity of truncated ABCA1 was recapitulated in an in vitro model using transfected Cos-7 cells. Our results suggest that the severe reduction of HDL-C in patients with truncation mutations may be at least partly explained by dominant negative suppression of expression and activity of the remaining full-length ABCA1 allele. These data suggest that ABCA1 requires a physical association with itself or other molecules for normal function and has important pharmacogenetic implications for individuals with truncation mutations.

A synthetic method for specific para -hydroxylation of nitroarenes has been developed. The reaction of nitronaphthalenes with cumeme hydroperoxide in basic aqueous media produces exclusively para -hydroxy nitronaphthalenes in good yield. The selectivity of ortho and para hydroxylation is mediated by water content. The rationale for water-controlled orientation of hydroxylation has been briefly discussed.

A new method for the synthesis of 2-alkyl- and 2-phenyl-5-hydrazinopyridine has been developed. Nucleophilic addition on pyridine by alkyl or phenyl lithium reagents generated a 2-substituted dihydropyridine anion that reacts with di- t -butyl azodicarboxylate to form 2,5-disubstituted dihydropyridine. Conversion of the dihydropyridines to pyridines was achieved by a mild air oxidation.

Traditional syntheses of benzodiazepines involve a Bischler-Napieralski reaction which is a three step process and gives low overall yields. An attractive alternative is to construct the diazepine ring under Pictet-Spengler conditions. This paper reports the synthesis of a novel pyridoindolo-benzodiazepine as a potent atypical antipsychotic agent. The key step in the synthesis is the ring formation of the diazepine ring in neat trifluoroacetic acid.

A general and effective synthesis of cyclic pentapeptides containing Arg-Gly-Asp sequence, which are potent antithrombotic agents, is reported.

Losoxantrone, a potent anticancer agent, has been efficiently synthesized in a 6 step process from 5-hydroxy-1,4-dichloroanthracene-9,10-dione.

Cytochrome P450c17 is the single enzyme having steroid 17 alpha-hydroxylase and 17,20 lyase activities. We sought to model the active site of this enzyme to identify residues contributing to its catalytic activities, and to test the roles of the identified amino acids by altering them via site-directed mutagenesis. Using the MIDAS-plus program, we modeled P450c17 and the structurally related steroid 21-hydroxylase, P450c21, on the crystallographically determined structure of bacterial P450cam. By positioning the progesterone substrate into each model, we identified five residues that appeared crucial for determining whether progesterone would undergo 17 alpha-hydroxylation (by P450c17) or 21-hydroxylation (by P450c21). Each identified residue in the P450c17 sequence was changed to the corresponding residue in the P450c21 sequence, yielding the four P450c17 mutants L102Y, G111D, G301l, and M369L + l371L. The mutants were transfected into COS-1 cells and their 17 alpha-hydroxylase, 17,20 lyase, and 21-hydroxylase activities were assayed by incubation with [14C]pregnenolone, [3H]17OH-pregnenolone, and [3H]17OH-progesterone and TLC. The L102Y and M369L + l371L mutants retained 50-80% of 17 alpha-hydroxylase and 70-100% of 17,20 lyase activity, while the G111D and G301l mutants lost both activities, but no mutants acquired detectable 21-hydroxylase activity (0.1% of wild type P450c21). Combination of the two mutants that retained partial activity (L102Y and M369L + l371L) yielded a single protein that retained 40% of 17 alpha-hydroxylase and 50% of 17,20 lyase activity, but none of the seven possible vectors expressing two, three, or all four of the mutations in a single enzyme yielded detectable 21-hydroxylase activity. The mutations D298V and D298S were predicted to ablate 17,20 lyase activity while retaining 17 alpha-hydroxylase activity, but were both inactive. These studies indicate that models based on the crystal structure of P450cam correctly predict many gross architectural features of steroidogenic enzymes and that many of the predicted residues are in or near the active site of P450c17. However, because enzymatic activity requires interactions between the enzyme and substrate at distances of less than 1 A, and modeling cannot predict atomic loci to greater than 1.5-2.0 A, it was not possible to design mutants that would confer 21-hydroxylase activity to P450c17. Currently available data cannot predict the structural and amino acid sequence requirements for a specific P450 activity.

17 alpha-Hydroxylase deficiency blocks the biosynthesis of cortisol and sex steroids, resulting in mineralocorticoid excess, hypertension, sexual infantilism, and female phenotype in both genetic sexes. The disease is caused by mutations in the gene encoding cytochrome P450c17, which is the single enzyme that mediates both 17 alpha-hydroxylase and 17,20-lyase activities. We report a 14-yr-old patient from Thailand with a classical clinical presentation of this rare disorder. Analysis of her P450c17 gene by polymerase chain reaction amplification and sequencing showed a nine-base deletion, eliminating codons 487-489 (Asp-Ser-Phe) near the carboxy-terminus of P450c17. This deletion creates a BclI site in the mutant DNA, permitting accurate demonstration that the patient was homozygous for this lesion, whereas one parent and two siblings were heterozygous. By use of site-directed mutagenesis, we created a vector that could express this mutated form of P450c17 when transfected into non-steroidogenic COS-1 cells. Such transfected cells produced immunodetectable P450c17 protein, but had no 17 alpha-hydroxylase or 17,20-lyase activity, whereas cells similarly transfected with a vector expressing normal human P450c17 could 17 alpha-hydroxylate either pregnenolone or progesterone and convert 17 alpha-hydroxypregnenolone to dehydroepiandrosterone, showing the presence of both activities. This is the first report of the molecular genetic basis of 17 alpha-hydroxylase deficiency in a Southeast Asian patient.

Pioglitazone, a hypoglycemic agent, has been shown to increase plasma HDL cholesterol, but the mechanism is incompletely understood. We further investigated effects of pioglitazone on transcriptional regulation of apolipoprotein (apo)A-I gene and functional properties of pioglitazone-induced apoA-I-containing particles. Pioglitazone dose-dependently stimulated apoA-I promoter activities in HepG2 cells. A peroxisome proliferator-activated receptor (PPAR)-response element located in site A (-214 to -192 bp, upstream of the transcription start site) of the promoter is required for pioglitazone-induced apoA-I gene transcription. Deletion of site A (-214 to -192 bp), B (-169 to -146 bp), or C (-134 to -119 bp), which clusters a number of cis-acting elements for binding of different transcription factors, reduced the basal apoA-I promoter activities, and no additional pioglitazone-sensitive elements were found within this region. Overexpression or knock-down of liver receptor homolog-1, a newly identified nuclear factor with strong stimulatory effect on apoA-I transcription, did not alter pioglitazone-induced apoA-I transcription. Pioglitazone-induced apoA-I transcription is mainly mediated through PPARalpha but not PPARgamma in hepatocytes. Pioglitazone induced production of HDL enriched in its subfraction containing apoA-I without apoA-II, which inhibited monocyte adhesion to endothelial cells in vitro. In conclusion, pioglitazone increases apoA-I production by directly enhancing PPAR-response element-dependent transcription, resulting in generation of apoA-I-containing HDL particles with increased anti-inflammatory property.

The Wisconsin hypoalpha mutant (WHAM) chicken has a >90% reduction in plasma HDL due to hypercatabolism. by the kidney of lipid-poor apoA-I. The WHAM chickens have a recessive white skin phenotype caused by a single-gene mutation that maps to the chicken Z-chromosome. This corresponds to human 9q31.1, a chromosomal segment that contains the ATP-binding cassette protein-1 (ABCA1) gene, which is mutated in Tangier Disease and familial hypoalphalipoproteinemia. Complete sequencing of the WHAM ABCA1 cDNA identified a missense mutation near the N-terminus of the protein (E89K). The substitution of this evolutionary conserved glutamate residue for lysine in the mouse ABCA1 transporter leads to complete loss of function, resulting principally from defective intracellular trafficking and very little ABCA1 reaching the plasma membrane. The WHAM chicken is a naturally occurring animal model for Tangier Disease.-Attie, A. D., Y. Hamon, A. R. Brooks-Wilson, M. P. Gray-Keller, M. L. E. MacDonald, V. Rigot, A. Tebon, L.-H. Zhang, J. D. Mulligan, R. R. Singaraja J.J. Bitgood, M. E. Cook, J.J. P. Kastelein, G. Chimini, and M. R. Hayden. Identification and functional analysis of a naturally occurring E89K mutation in the ABCA1 gene of the WHAM chicken

ATP-binding cassette A1 (ABCA1) is a key mediator of cholesterol and phospholipid efflux to apolipoprotein particles. We show that ABCA1 is a constitutively phosphorylated protein in both RAW macrophages and in a human embryonic kidney cell line expressing ABCA1. Furthermore, we demonstrate that phosphorylation of ABCA1 is mediated by protein kinase A (PKA) or a PKA-like kinase in vivo. Through site-directed mutagenesis studies of consensus PKA phosphorylation sites and in vitro PKA kinase assays, we show that Ser-1042 and Ser-2054, located in the nucleotide binding domains of ABCA1, are major phosphorylation sites for PKA. ApoA-I-dependent phospholipid efflux was decreased significantly by mutation of Ser-2054 alone and Ser-1042/Ser-2054 but was not significantly impaired with Ser-1042 alone. The mechanism by which ABCA1 phosphorylation affected ApoA-I-dependent phospholipid efflux did not involve either alterations in ApoA-I binding or changes in ABCA1 protein stability. These studies demonstrate a novel serine (Ser-2054) on the ABCA1 protein crucial for PKA phosphorylation and for regulation of ABCA1 transporter activity.

By using BAC transgenic mice, we have shown that increased human ABCA1 protein expression results in a significant increase in cholesterol efflux in different tissues and marked elevation in high density lipoprotein (HDL)-cholesterol levels associated with increases in apoAI and apoAII. Three novel ABCA1 transcripts containing three different transcription initiation sites that utilize sequences in intron 1 have been identified. In BAC transgenic mice there is an increased expression of ABCA1 protein, but the distribution of the ABCA1 product in different cells remains similar to wild type mice. An internal promoter in human intron 1 containing liver X response elements is functional in vivo and directly contributes to regulation of the human ABCA1 gene in multiple tissues and to raised HDL cholesterol, apoAI, and apoAII levels. A highly significant relationship between raised protein levels, increased efflux, and level of HDL elevation is evident. These data provide proof of the principle that increased human ABCA1 efflux activity is associated with an increase in HDL levels in vivo.

We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver. The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.