Molecular chaperones of the Hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. Target peptides were labeled with an environmentally sensitive fluorophore so that their binding to the molecular chaperone DnaK of Escherichia coli could be followed in real time. The two-step process was characterized by relaxation times of 27 seconds and 200 seconds with 2 microM DnaK and 0.1 microM ligand at 25 degrees C. In the presence of adenosine triphosphate, the formation of the complex was greatly accelerated and appeared to be a single-exponential process with a relaxation time of 0.4 second. The binding-release cycle of DnaK thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the adenosine triphosphatase activity of the chaperone (turnover number, 0.13 per minute at 30 degrees C).

Botulinum-A toxin injections into the detrusor seem to be a safe and valuable therapeutic option in spinal cord injured patients with incontinence resistant to anticholinergic medication who perform clean intermittent self-catheterization. Successfully treated patients become continent again and may withdraw from or markedly decrease anticholinergic drug intake. A dose of 300 units botulinum-A toxin seems to be needed to counteract an overactive detrusor. The duration of bladder paresis induced by the toxin is at least 9 months, when repeat injections are required.

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.

Hydrolytic deamination of 5-methylcytosine leads to the formation of G/T mismatches. We have shown previously that these G/T mispairs are corrected to G/C pairs by a mismatch-specific thymine-DNA glycosylase, TDG, which we subsequently purified from human cells. Here we describe the cloning of the human cDNA encoding TDG. We have identified two distinct cDNA species that differ by 100 nucleotides at the 3-untranslated region. These cDNAs contain a 410-amino acid open reading frame that encodes a 46-kDa polypeptide. The G/T glycosylase, expressed both in vitro and in Escherichia coli, migrated in denaturing polyacrylamide gels with an apparent size of 60 kDa. The substrate specificity of the recombinant protein corresponded to that of the cellular enzyme, and polyclonal antisera raised against the recombinant protein neutralized both activities. We therefore conclude that the cDNA described below encodes human TDG. Data base searches identified a serendipitously cloned mouse cDNA sequence that could be shown to encode the murine TDG homologue. No common amino acid sequence motifs between the G/T-specific enzyme and other DNA glycosylases could be found, suggesting that TDG belongs to a new class of baseexcision repair enzymes.

Our results show that intradetrusor botulinum-A toxin injections may be an efficient and safe treatment option in patients with severe overactive bladder resistant to all conventional treatments.

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