Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21

GM2/GD2 synthase gene knockout mice lack all complex gangliosides, which are abundantly expressed in the nervous systems of vertebrates. In turn, they have increased precursor structures GM3 and GD3, probably replacing the roles of the depleted complex gangliosides. In this study, we found that 9-O-acetyl GD3 is also highly expressed as one of the major glycosphingolipids accumulating in the nervous tissues of the mutant mice. The identity of the novel component was confirmed by neuraminidase treatment, thin layer chromatography-immunostaining, two-dimensional thin layer chromatography with base treatment, and mass spectrometry. All candidate factors reported to be possible inducer of 9-O- acetylation, such as bitamine D binding protein, acetyl CoA transporter, or O-acetyl ganglioside synthase were not up-regulated. Tis21 which had been reported to be a 9-O-acetylation inducer was partially down-regulated in the null mutants, suggesting that Tis21 is not involved in the induction of 9-O-acetyl-GD3 and that accumulated high amount of GD3 might be the main factor for the dramatic increase of 9-O-acetyl GD3. The ability to acetylate exogenously added GD3 in the normal mouse astrocytes was examined, showing that the wild-type brain might be able to synthesize very low levels of 9-O-acetyl GD3. Increased 9-O-acetyl GD3, in addition to GM3 and GD3, may play an important role in the compensation for deleted complex gangliosides in the mutant mice.     

Investigation of coenzyme Q biosynthesis in human fibroblast and HepG2 cells

Coenzyme Q (CoQ) deficiency occurs in genetic disorders, during aging and various diseases. Diagnosis requires skin fibroblasts in tissue culture. [³H]Mevalonate incorporation was appropriate to measure the rate of CoQ synthesis in fibroblasts and hepatoblastoma cells. [¹⁴C]p-Hydroxybenzoate had limited permeability, but it could be increased with Fugene and cyclodextrin. Inhibition of decaprenyl-4-hydroxybenzoate transferase results in the accumulation of decaprenyl diphosphate, an indicator of enzyme deficiency. Also, analysis of the corresponding mRNAs in this case is useful. In vitro assays to measure trans-prenyltransferase and decaprenyl-4-hydroxybenzoate transferase activities are not available. Neither measurement of methyltransferases is reliable in human cells. In vitro reconstruction of CoQ synthesis, in opposite to cholesterol synthesis, proved to be unsuccessful. Thus, the biochemical characterization of the CoQ biosynthetic system in human cells is restricted to a few reliable analytical procedures.     

A member of the maize isopentenyl transferase gene family, <Emphasis Type="Italic">Zea mays isopentenyl transferase 2</Emphasis> (<Emphasis Type="Italic">ZmIPT2</Emphasis>), encodes a cytokinin biosynthetic enzyme expressed during kernel development

Cytokinins (CKs) are plant hormones that regulate a large number of processes associated with plant growth and development such as induction of stomata opening, delayed senescence, suppression of auxin-induced apical dominance, signaling of nitrogen availability, differentiation of plastids and control of sink strength. In maize, CKs are thought to play an important role in establishing seed size and increasing seed set under normal and unfavorable environmental conditions therefore influencing yield. In recent years, the discovery of isopentenyl transferase (IPT) genes in plants has shed light on the CK biosynthesis pathway in plants. In an effort to increase our understanding of the role played by CKs in maize development and sink-strength, we identified several putative IPT genes using a bioinformatics approach. We focused our attention on one gene in particular, ZmIPT2, because of its strong expression in developing kernels. The expression of the gene and its product overlays the change in CK levels in developing kernels suggesting a major role in CK biosynthesis for kernel development. We demonstrate that at 8–10 days after pollination (DAP) the endosperm and especially the basal transfer cell layer (BETL) is a major site of ZmIPT2 expression, and that this expression persists in the BETL and the developing embryo into later kernel development stages. We show that ectopic expression of ZmIPT2 in calli and in planta created phenotypes consistent with CK overproduction. We also show that ZmIPT2 preferentially uses ADP and ATP over AMP as the substrates for dimethylallyl diphosphate (DMAPP) IPT activity. The expression pattern of ZmIPT2 in the BETL, endosperm and embryo during kernel development will be discussed with an emphasis on the suggested role of CKs in determining sink-strength and grain production in crop plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of putative hydrophobic substrate binding site residues of a Delta class glutathione transferase from Anopheles dirus

To date, investigations of the hydrophobic substrate site of the insect Delta class glutathione transferase are limited in number. In the present study, putative hydrophobic site residues of AdGSTD4-4 have been proposed and characterized. These residues are Gln-112, Thr-174, Phe-212, Arg-214, Tyr-215 and Phe-216. It was found that Gln-112 does not contribute significantly to the catalytic properties of AdGSTD4-4. Arg-214, Tyr-215 and Phe-216 made contributions to catalytic properties and the rate-limiting step. Thr-174 and Phe-212 appeared to be important in enzymatic catalysis by stabilizing the active site β1-α1 loop on which the critical catalytic residue Ser-9 is located. The aromatic Phe-212 pi cloud appears to be important for interactions with its hydrophobic size representing an almost equally important factor. The data suggests that these residues are not directly involved in catalysis but exert their influence through secondary interactions. In addition, active site rearrangements occur to bring different residues into play even for conjugation through the same mechanisms. Therefore, due to the conformational rearrangements topologically equivalent residues observed in crystal structures may not perform equivalent roles in catalysis in different GST classes.     

Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 °C.     

Low Night Temperature-Induced Changes in Photosynthesis and Rubber Accumulation in Guayule (Parthenium Argentatum Gray)

Three-year-old plants of Parthenium argentatum Gray cv. 11591 grown under natural photoperiod were exposed for 60 d to low night temperature (LNT) of 15 °C (daily from 18:00 to 06:00). Effects of the treatment on net photosynthetic rates (P _N), rubber accumulation, and associated biochemical traits were examined. LNT initially reduced P _N with a parallel decline in the activities of ribulose-1,5-bisphosphate carboxylase, fructose bisphosphatase, and sucrose phosphate synthase for 20–30 d. Later, LNT enhanced P _N and the activities of photosynthetic enzymes. Associated with high P _N in LNT-treated guayule plants was a two-fold increase in rubber content and rubber transferase activity per unit of protein. The initial decrease in P _N in LNT-treated guayule was associated with low content of chlorophyll (a+b), large starch accumulation, and higher ratio of glucose-6-phosphate/fructose-6-phosphate. Photosystem 2 activity in isolated chloroplasts was initially decreased, but increased after 30 d. There was a significant increase in the leaf soluble protein content in LNT-treated plants. Hence the photosynthetic performance of plants grown at 15 °C night temperature for 50 d was superior to those grown under natural photoperiod in all parameters studied. The high photosynthetic capacity may contribute to superior rubber yields under LNT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Association between COMT SNP variation and timidity in Golden and Labrador Retrievers

Timidity in dogs is a trait with high heritability. However, the relevant genetic factors and markers associated with this condition are largely unknown. The function of the catechol‐O‐methyl transferase (COMT) gene has been found to be associated with human fearful or anxious emotions, and the COMT:p.Val158Met polymorphism locus is significantly related to anxious behavior. In the present study, the correlation between timidity and four single nucleotide polymorphism (SNP) variations (C.‐1666C>G c.39A>G, c.216G>A, c.482G>A) of the COMT gene was investigated in dogs. The evaluation was based on the dog courage assessment assay and a genotype and haplotype analysis in Labrador Retrievers (LR) and Golden Retrievers (GR). The principal components analysis factor structure of the courage phenotype was invariant between LR and GR. Sex, breed and age had no statistically significant effect on the timidity of the dogs. All SNP loci detected were in Hardy–Weinberg equilibrium. The c.39A>G locus was removed in the subsequent association analysis due to the significant difference between LR and GR in genotype distributions. Intriguingly, the genotypes and haplotypes of the COMT gene were significantly and highly correlated with the timidity of LR and GR. The A alleles of the COMT:c.216G>A and c.482G>A loci appeared to play a dominant role in the timid behavior of the dogs. This result supports and broadens the warrior/worrier hypothesis and will have important implications for the understanding of the evolution of temperament in dogs. Additionally, the results provide predictive genetic markers for temperament in dogs.