Polyunsaturated fatty acids suppress sterol regulatory element-binding protein 1c promoter activity by inhibition of liver X receptor (LXR) binding to LXR response elements.

Previous studies have demonstrated that polyunsaturated fatty acids (PUFAs) suppress sterol regulatory element-binding protein 1c (SREBP-1c) expression and, thus, lipogenesis. In the current study, the molecular mechanism for this suppressive effect was investigated with luciferase reporter gene assays using the SREBP-1c promoter in HEK293 cells. Consistent with previous data, the addition of PUFAs to the medium in the assays robustly inhibited the SREBP-1c promoter activity. Deletion and mutation of the two liver X receptor (LXR)-responsive elements (LXREs) in the SREBP-1c promoter region eliminated this suppressive effect, indicating that both LXREs are important PUFA-suppressive elements. The luciferase activities of both SREBP-1c promoter and LXRE enhancer constructs induced by co-expression of LXRalpha or -beta were strongly suppressed by the addition of various PUFAs (arachidonic acid > eicosapentaenoic acid > docosahexaenoic acid > linoleic acid), whereas saturated or mono-unsaturated fatty acids had minimal effects. Gel shift mobility and ligand binding domain activation assays demonstrated that PUFA suppression of SREBP-1c expression is mediated through its competition with LXR ligand in the activation of the ligand binding domain of LXR, thereby inhibiting binding of LXR/retinoid X receptor heterodimer to the LXREs in the SREBP-1c promoter. These data suggest that PUFAs could be deeply involved in nutritional regulation of cellular fatty acid levels by inhibiting an LXR-SREBP-1c system crucial for lipogenesis.     

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.     

Cumulative effect of amino acid replacements results in enhanced thermostability of potato type L alpha-glucan phosphorylase

The thermostability of potato type L alpha-glucan phosphorylase (EC 2.4.1.1) was enhanced by random and site-directed mutagenesis. We obtained three single-residue mutations-Phe39-->Leu (F39L), Asn135-->Ser (N135S), and Thr706-->Ile (T706I)-by random mutagenesis. Although the wild-type enzyme was completely inactivated, these mutant enzymes retained their activity even after heat treatment at 60 degrees C for 2 h. Combinations of these mutations were introduced by site-directed mutagenesis. The simultaneous mutation of two (F39L/N135S, F39L/T706I, and N135S/T706I) or three (F39L/N135S/T706I) residues further increased the thermostability of the enzyme, indicating that the effect of the replacement of the residues was cumulative. The triple-mutant enzyme, F39L/N135S/T706I, retained 50% of its original activity after heat treatment at 65 degrees C for 20 min. Further analysis indicated that enzymes with a F39L or T706I mutation were resistant to possible proteolytic degradation.     

Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids

We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.     

Nucleosides and nucleotides. 126. Incorporation of a mutagenic nucleoside, 5-formyl-2′-deoxyuridine, into an oligodeoxyribonucleotide

An oligodeoxyribonucleotide containing 5-formyl-2′-deoxyuridine (1) was synthesized using a new protecting group of the formyl group.     

Stabilization of trypsin by modification with comb-shaped copolymers of poly(ethylene glycol) derivative and maleic anhydride

Summary Trypsin from bovine pancreas was coupled with copolymers of poly(ethylene glycol) derivative and maleic anhydride with the molecular weights of 13 kDa and 100 kDa (activated PM₁₃ and PM₁₀₀). The modified trypsins were more stable towards autolysis and heat- or urea-treatment than nonmodified trypsin. Stabilization of trypsin caused by the chemical modification with activated PMs is discussed in relation to the protein conformation.     

Molecular analysis of the recombination junctions of lambda bio transducing phages.

Summary To examine the mechanism of recombination involved in the formation of specialized transducing phage during the induction of bacteriophage we have determined the nucleotide sequences of the recombination junctions of bio phages. The results indicate that abnormal excision takes place at many sites on both bacterial and phage genomes and that the recombination sites have short regions of homology (5–14 bp). Some of the sequences of the recombination sites were similar to the consensus sequences of DNA gyrase-cleavage sites and repetitive extragenic palindromic (REP) sequences. These results showed that abnormal excision is a type of illegitimate recombination. The possible involvement of DNA gyrase in this recombination is discussed.     

Late-Phase Accumulation of Inositol Phosphates Stimulated by Prostaglandins D2 and F2α in Neuroblastoma × Glioma Hybrid NG108-15 Cells

The accumulation of inositol phosphates (IPs) in response to prostaglandins (PGs) was studied in NG108-15 cells preincubated with myo-[3H]inositol. As a positive control, bradykinin caused accumulation of IPs transiently at an early phase (within 1 min) and continuously during a late phase (15-60 min) of incubation in the cells. PGD2 and PGF2 alpha did not significantly cause the accumulation of IPs at an early phase but significantly stimulated inositol bisphosphate (IP2) and inositol monophosphate (IP) formation at late phase of incubation. The maximum stimulation was obtained at greater than 10(-7) M concentrations of these PGs, the levels being three-and twofold for IP2 and IP1, respectively. 9 alpha, 11 beta-PGF2 has a slight effect but PGE2 and the metabolites of PGD2 and PGF2 alpha have no effect up to 10(-6)M. The effects of PGD2 and PGF2 alpha were not additive, but the effect of each PG was additive to that of bradykinin at a late phase of incubation. Inositol 1-monophosphate was mainly identified in the stimulation by 10(-5) M PGD2 and 10(-5) M PGF2 alpha, whereas both inositol 1-monophosphate and inositol 4-monophosphate were produced in the stimulation by 10(5) M bradykinin. Depletion of extracellular Ca2+ diminished the stimulatory effect of PGD2 and PGF2 alpha and late-phase effect of bradykinin, but simple Ca2+ influx into the cells by high K+, ionomycin, or A23187 failed to cause such late-phase effects. These results suggest that PGD2 and PGF2 alpha specifically stimulate hydrolysis of inositol phospholipids.     

Ester-exchange catalyzed by lipase modified with polyethylene glycol

Lipoprotein lipase was modified with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine; forty-six percent out of seven amino groups in the molecule were substituted. The modified lipase catalyzed ester-exchange reactions between an ester and an alcohol, between an ester and an acid, and between two esters. The modified enzyme catalyzed these reactions not only in organic solvents, but also in straight hydrophobic substrates. As the modified enzyme was extremely stable at elevated temperature, for example at 70 degrees C, this can find many practical applications.