Genetic Analysis of Central Carbon Metabolism Unveils an Amino Acid Substitution That Alters Maize NAD-Dependent Isocitrate Dehydrogenase Activity

Background

Central carbon metabolism (CCM) is a fundamental component of life. The participating genes and enzymes are thought to be structurally and functionally conserved across and within species. Association mapping utilizes a rich history of mutation and recombination to achieve high resolution mapping. Therefore, applying association mapping in maize (Zea mays ssp. mays), the most diverse model crop species, to study the genetics of CCM is a particularly attractive system.

Methodology/Principal Findings

We used a maize diversity panel to test the CCM functional conservation. We found heritable variation in enzyme activity for every enzyme tested. One of these enzymes was the NAD-dependent isocitrate dehydrogenase (IDH, E.C. 1.1.1.41), in which we identified a novel amino-acid substitution in a phylogenetically conserved site. Using candidate gene association mapping, we identified that this non-synonymous polymorphism was associated with IDH activity variation. The proposed mechanism for the IDH activity variation includes additional components regulating protein level. With the comparison of sequences from maize and teosinte (Zea mays ssp. Parviglumis), the maize wild ancestor, we found that some CCM genes had also been targeted for selection during maize domestication.

Conclusions/Significance

Our results demonstrate the efficacy of association mapping for dissecting natural variation in primary metabolic pathways. The considerable genetic diversity observed in maize CCM genes underlies heritable phenotypic variation in enzyme activities and can be useful to identify putative functional sites.     

Alcohol-induced autophagy contributes to loss in skeletal muscle mass

Patients with alcoholic cirrhosis and hepatitis have severe muscle loss. Since ethanol impairs skeletal muscle protein synthesis but does not increase ubiquitin proteasome-mediated proteolysis, we investigated whether alcohol-induced autophagy contributes to muscle loss. Autophagy induction was studied in: A) Human skeletal muscle biopsies from alcoholic cirrhotics and controls, B) Gastrocnemius muscle from ethanol and pair-fed mice, and C) Ethanol-exposed murine C2C12 myotubes, by examining the expression of autophagy markers assessed by immunoblotting and real-time PCR. Expression of autophagy genes and markers were increased in skeletal muscle from humans and ethanol-fed mice, and in myotubes following ethanol exposure. Importantly, pulse-chase experiments showed suppression of myotube proteolysis upon ethanol-treatment with the autophagy inhibitor, 3-methyladenine (3MA) and not by MG132, a proteasome inhibitor. Correspondingly, ethanol-treated C2C12 myotubes stably expressing GFP-LC3B showed increased autophagy flux as measured by accumulation of GFP-LC3B vesicles with confocal microscopy. The ethanol-induced increase in LC3B lipidation was reversed upon knockdown of Atg7, a critical autophagy gene and was associated with reversal of the ethanol-induced decrease in myotube diameter. Consistently, CT image analysis of muscle area in alcoholic cirrhotics was significantly reduced compared with control subjects. In order to determine whether ethanol per se or its metabolic product, acetaldehyde, stimulates autophagy, C2C12 myotubes were treated with ethanol in the presence of the alcohol dehydrogenase inhibitor (4-methylpyrazole) or the acetaldehyde dehydrogenase inhibitor (cyanamide). LC3B lipidation increased with acetaldehyde treatment and increased further with the addition of cyanamide. We conclude that muscle autophagy is increased by ethanol exposure and contributes to sarcopenia.     

Gut Hormone Pharmacology of a Novel GPR119 Agonist (GSK1292263), Metformin,and Sitagliptin in Type 2 Diabetes Mellitus: Results from Two Randomized Studies

GPR119 receptor agonists improve glucose metabolism and alter gut hormone profiles in animal models and healthy subjects. We therefore investigated the pharmacology of GSK1292263 (GSK263), a selective GPR119 agonist, in two randomized, placebo-controlled studies that enrolled subjects with type 2 diabetes. Study 1 had drug-naive subjects or subjects who had stopped their diabetic medications, and Study 2 had subjects taking metformin. GSK263 was administered as single (25–800 mg; n = 45) or multiple doses (100–600 mg/day for 14 days; n = 96). Placebo and sitagliptin 100 mg/day were administered as comparators. In Study 1, sitagliptin was co-administered with GSK263 or placebo on Day 14 of dosing. Oral glucose and meal challenges were used to assess the effects on plasma glucose, insulin, C-peptide, glucagon, peptide tyrosine-tyrosine (PYY), glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). After 13 days of dosing, GSK263 significantly increased plasma total PYY levels by ∼five-fold compared with placebo, reaching peak concentrations of ∼50 pM after each of the three standardized meals with the 300 mg BID dose. Co-dosing of GSK263 and metformin augmented peak concentrations to ∼100 pM at lunchtime. GSK263 had no effect on active or total GLP-1 or GIP, but co-dosing with metformin increased post-prandial total GLP-1, with little effect on active GLP-1. Sitagliptin increased active GLP-1, but caused a profound suppression of total PYY, GLP-1, and GIP when dosed alone or with GSK263. This suppression of peptides was reduced when sitagliptin was co-dosed with metformin. GSK263 had no significant effect on circulating glucose, insulin, C-peptide or glucagon levels. We conclude that GSK263 did not improve glucose control in type 2 diabetics, but it had profound effects on circulating PYY. The gut hormone effects of this GPR119 agonist were modulated when co-dosed with metformin and sitagliptin. Metformin may modulate negative feedback loops controlling the secretion of enteroendocrine peptides.

Trial Registration:

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01119846","term_id":"NCT01119846"}}NCT01119846 Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01128621","term_id":"NCT01128621"}}NCT01128621     

Amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor)

The amino acid sequence of the heavy chain of human alpha-factor XIIa (activated Hageman factor) was determined by automated Edman degradation using the peptides produced by chemical and enzymatic cleavages of intact factor XII and alpha-factor XIIa. Combining this sequence with the previously determined sequence of beta-factor XIIa (Fujikawa, K., and McMullen, B. A. (1983) J. Biol. Chem. 258, 10924-10933), the complete amino acid sequence of human factor XII has been established. The heavy chain of alpha-factor XIIa is composed of 353 amino acid residues containing one Asn-linked and six probable O-linked carbohydrate chains. The heavy chain of alpha-factor XIIa appears to contain four different domains including a "kringle," a "growth factor" domain, and the "type I" and "type II" domains of fibronectin. The domain organization of factor XII is analogous to those of several fibrinolytic proteins, including tissue plasminogen activator and urokinase, suggesting that factor XII belongs to the same protease subfamily as these two proteins.     

Chemical synthesis of a mitochondrial gene designed for expression in the yeast nucleus

A novel DNA sequence coding for subunit 8 of the mitochondrial ATPase of Saccharomyces cerevisiae has been constructed by chemical synthesis. The synthetic gene, termed NAP1, is designed for expression in the yeast nucleus and codes for a 48 amino acid polypeptide identical to that encoded by the mitochondrial aap1 gene of S. cerevisiae. The codons chosen for the NAP1 sequence correspond almost exclusively to those most frequently occurring in highly expressed yeast genes. The NAP1 coding region differs in 31 codons from that of aap1, and is flanked by sequences carrying restriction enzyme sites useful for cloning and for gene expression. A 170 bp double stranded DNA molecule was constructed by assembling 12 oligonucleotides (12 to 45 bases in length) in a single annealing/ligation mixture. This synthetic gene will provide a route for the systematic manipulation, through in vitro mutagenesis, of the structure of a protein normally encoded by mitochondrial DNA.     

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.     

Transformation of 12 different plasmids into soybean via particle bombardment

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.