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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)
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)
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)
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)
The refolding of ribonuclease T1 is dominated by two major slow kinetic phases that show properties of proline isomerization reactions. We report here that the molecular origin of one of these processes is the trans----cis isomerization of the Ser54-Pro55 peptide bond, which is cis in the native protein but predominantly trans in unfolded ribonuclease T1.(More)
It is our aim to elucidate molecular aspects of the mechanism of protein folding. We use ribonuclease T1 as a model protein, because it is a small single-domain protein with a well-defined secondary and tertiary structure, which is stable in the presence and absence of disulfide bonds. Also, an efficient mutagenesis system is available to produce protein(More)
De novo designed signal peptidase I cleavage sites were tested for their biological activity in vivo in an Escherichia coli expression and secretion system. The artificial cleavage site sequences were generated by two different computer-based design techniques, a simple statistical method, and a neural network approach. In previous experiments, a neural(More)
Intense radiation from lasers has opened up many new areas of research in physics and chemistry, and has revolutionized optical technology. So far, most work in the field of nonlinear processes has been restricted to infrared, visible and ultraviolet light, although progress in the development of X-ray lasers has been made recently. With the advent of a(More)