A Leucine-to-Proline Substitution Causes a Defective α-Antichymotrypsin Allele Associated with Familial Obstructive Lung Disease

Using denaturing gradient gel electrophoresis and direct sequencing of amplified genomic DNA, we have identified two defective mutants of the human α₁-antichymotrypsin (ACT) gene associated with chronic obstructive pulmonary disease (COPD). A leucine 55-to-proline substitution causing a defective ACT allele (Bochum-1) was observed in a family with COPD in three subsequent generations. Another mutation, proline 229-to-alanine (Bonn-1), was associated with ACT serum deficiency in four patients with a positive family history. These mutations were, not detected among 100 healthy control subjects, suggesting a possible pathogenetic role of ACT gene defects in a subset of patients with COPD.     

Die wirkung von spindelgiften aufLupinus luteus L.

Ohne Zusammenfassungmit 6 AbbildungenHerrn Prof. Dr. R. Bauch zum 60. Geburtstag gewidmet.     

Coevolution of Bacteriophage PP01 and Escherichia coli O157:H7 in Continuous Culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h⁻¹ and by 3 orders of magnitude at a lower dilution rate (0.327 h⁻¹). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h⁻¹ and persisted until the end of the experiment (~200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.     

A cyclophilin-like peptidyl-prolyl cis/trans isomerase from Legionella pneumophila – characterization, molecular cloning and overexpression

Legionella pneumophila is the causative agent of a severe form of pneumonia in humans (Legionnaires’disease). A major virulence factor, the Mip protein (FK506-binding protein, FKBP25mem), belongs to the enzyme family of peptidyl-prolyl cis/trans isomerases (PPIases). Here we show that L. pneumophila Philadelphia I possesses an additional cytoplasmic PPiase at a level of enzyme activity comparable to that of FKBP25mem. The N-terminal amino acid sequence of the purified protein was obtained by Edman degradation and showed that the protein is a member of the cyclophilin family of PPIases. The Icy gene (Legionella cycophn) was cloned and sequenced. It encodes a putative 164-amino-acid protein with a molecular mass of 17 968 Da called L. pneumophila cyclophilin 18 (L. p. Cyp18). Amino acid sequence comparison displays considerable similarity to the cytoplasmic and the periplasmic cyclophilins of Escherichia coll with 60.5% and 51.5% identity, respectively. The substrate specificity and inhibition by cyclosporin A revealed a pattern that is typically found for other bacterial cyclophilins. An L. pneumophila Cyp18 derivative with a 19-amino-acid polypeptide extension including a 6-histi-dine tag and an enterokinase cleavage site exhibits     

UDP-glucose is a potential intracellular signal molecule in the control of expression of sigma S and sigma S-dependent genes in Escherichia coli.

The sigma S subunit of RNA polymerase is the master regulator of a regulatory network that controls stationary-phase induction as well as osmotic regulation of many genes in Escherichia coli. In an attempt to identify additional regulatory components in this network, we have isolated Tn10 insertion mutations that in trans alter the expression of osmY and other sigma S-dependent genes. One of these mutations conferred glucose sensitivity and was localized in pgi (encoding phosphoglucose isomerase). pgi::Tn10 strains exhibit increased basal levels of expression of osmY and otsBA in exponentially growing cells and reduced osmotic inducibility of these genes. A similar phenotype was also observed for pgm and galU mutants, which are deficient in phosphoglucomutase and UDP-glucose pyrophosphorylase, respectively. This indicates that the observed effects on gene expression are related to the lack of UDP-glucose (or a derivative thereof), which is common to all three mutants. Mutants deficient in UDP-galactose epimerase (galE mutants) and trehalose-6-phosphate synthase (otsA mutants) do not exhibit such an effect on gene expression, and an mdoA mutant that is deficient in the first step of the synthesis of membrane-derived oligosaccharides, shows only a partial increase in the expression of osmY. We therefore propose that the cellular content of UDP-glucose serves as an internal signal that controls expression of osmY and other sigma S-dependent genes. In addition, we demonstrate that pgi, pgm, and galU mutants contain increased levels of sigma S during steady-state growth, indicating that UDP-glucose interferes with the expression of sigma S itself.     

Effect of Calcitriol in Combination with Corticosterone, Interleukin-1β, and Transforming Growth Factor-β1 on Nerve Growth Factor Secretion in an Astroglial Cell Line

Abstract: In astrocytes, nerve growth factor (NGF) synthesis has been described to be stimulated by the cytokines interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) and inhibited by corticosterone. As all three factors are present in the brain under certain conditions, we investigated the effect of their combined application on NGF secretion in the astroglial cell line RC7 and, in addition, studied the effect of calcitriol (1α,25-dihydroxyvitamin D₃). Calcitriol stimulated NGF secretion, whereas corticosterone reduced basal levels of NGF secretion as well as inhibited the NGF secretion induced by IL-1β, calcitriol, and TGF-β1. Calcitriol had an additive effect when applied together with IL-1β and a synergistic effect when applied with TGF-β1. Moreover, calcitriol not only counteracted the inhibitory effect of corticosterone on NGF secretion stimulated by TGF-β1 but even augmented it to a level more than threefold higher than that reached with TGF-β1 alone. Due to the trophic effect of NGF on basal forebrain cholinergic neurons, these findings might be of therapeutic relevance under conditions where cholinergic function is impaired and the endogenous levels of corticosterone, IL-1β, or TGF-β1 are elevated.