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We have developed a general and rapid method for site-directed mutagenesis using primed amplification by the polymerase chain reaction. Starting from a double-stranded DNA template, this method requires only one single specific mutagenic primer and two universal sequencing primers flanking the region to be mutated further upstream and downstream,(More)
The slow refolding of ribonuclease T1 was investigated by different probes. Structural intermediates with secondary structure are formed early during refolding, as indicated by the rapid regain of a native-like circular dichroism spectrum in the amide region. This extensive structure formation is much faster than the slow steps of refolding, which are(More)
We have studied the absorption and metabolism of resveratrol in the jejunum in an isolated rat small intestine model. Only small amounts of resveratrol were absorbed across the enterocytes of the jejunum and ileum unmetabolised. The major compound detected on the serosal side was the glucuronide conjugate of resveratrol (96.5% +/- 4.6 of the amount(More)
CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxylation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation and the interaction of hydrophobic substrates with soluble enzymes. Here the molecular cloning of the CYP153 gene is reported. Single(More)
The 60 kb circular mitochondrial genome of N. crassa has previously been shown to contain a single transcription unit for 17S and 24S rRNA mapping within the largest Eco RI fragment E1 (19.6 kb). This fragment was isolated from uncloned mitochondrial DNA and further analyzed by cleavage with restriction endonucleases Hind II, Hind III, Bam HI, Pvu II and(More)
A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart(More)
The purpose of this study was to investigate biomarkers of the bioavailability and metabolism of hydroxycinnamate derivatives through the determination of the pharmacokinetics of their urinary elimination and identification of the metabolites excreted. Coffee was used as a rich source of caffeic acid derivatives and human supplementation was undertaken. The(More)
There is considerable interest in the bioavailability of polyphenols and their bioactivity in vivo. We have studied the absorption and metabolism of catechin and epicatechin in the small intestine and the comparative transfer across the jejunum and ileum. Perfusion of isolated jejunum with the flavanols resulted in glucuronidation ( approximately 45%),(More)
For 30 years, the prevailing view has been that the hydrophobic effect contributes considerably more than hydrogen bonding to the conformational stability of globular proteins. The results and reasoning presented here suggest that hydrogen bonding and the hydrophobic effect make comparable contributions to the conformational stability of ribonuclease T1(More)
A consensus structure model of 5S rRNA presenting all conserved nucleotides in fixed positions has been deduced from the primary and secondary structure of 71 eubacterial, archaebacterial, eukaryotic cytosolic and organellar molecules. Phylogenetically related groups of molecules are characterized by nucleotide deletions in helices III, IV and V, and by(More)