Identification by in vitro complementation of regions required for cell-invasive activity of Bordetella pertussis adenylate cyclase toxin

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a 1706-residue protein composed of an amino-terminal adenylate cyclase (AC) domain linked to a 1300-residue channel-forming RTX ( r epeats in t o x in) haemolysin. The toxin delivers its AC domain into a variety of eukaryotic cells and impairs cellular functions by catalysing unregulated synthesis of cAMP from intracellular ATP. We have examined toxin activities of a set of deletion derivatives of CyaA. The results indicate that CyaA does not have a dedicated target cell-binding domain and that structural integrity and co-operation of all domains, as well as the post-translational fatty acylation mediated by an accessory protein CyaC, are all essential for target cell association and toxin activity of CyaA. When tested individually, all toxin derivatives were inactive and impaired in the tight association with the target cell surface. However, pairs of constructs with non-overlapping deletions complemented each other in vitro and exhibited a partially restored cytotoxic activity. This suggests that at least a part of the active toxin may act in the form of dimers or higher oligomers. The complementation analysis revealed that the last 217 residues of CyaA, containing the unprocessed secretion signal, form an autonomous domain essential for toxin activity, and that the region from residue 624 to 780 may be directly involved in delivery of the AC toxin into cells.     

Reversal of IAA-induced inhibition of flowering by aminoethoxyvinylglycine inChenopodium

Aminoethoxyvinylglycine (AVG) applied as a droplet (3 l, 0.1 mM) to the plumule of seedlings of both the short-day plantChenopodium rubrum and the long-day plantChenopodium murale counteracted to a great extent or even canceled the inhibition of flowering due to exogenous indole-3-acetic acid (IAA). This effect was more pronounced with the two substances administered simultaneously than with later application of AVG alone. AVG by itself in some cases promoted the percentage of flowering in bothChenopodium species. Application of IAA to the shoot apex was shown to elevate ethylene production in both species, whereas application of AVG alone was shown to suppress it. Thus, ethylene may be considered an active agent of flowering inhibition brought about by IAA application.     

Anomalous litters in hybrid mice and the retention of spermatozoa in the female tract.

Suspected superfetation was investigated in a Glasgow hybrid stock of mice. The male was removed either (i) a few days before parturition, or (ii) immediately after mating and on 23 and 25 occasions, respectively, a second litter was born. Members of the anomalous litters were inbred for 10 generations, but the incidence of supernumerary litters did not increase beyond 2-5%. The anterior part of over 500 reproductive tracts, at various stages of pregnancy and after parturition, were serially sectioned but a second set of embryos was not found. The second gestation was of normal length and superfetation was not therefore considered to be the cause of the anomalous litters. In two females, one non-pregnant and one pregnant, spermatozoa were found in the uterus and oviducts 8 days after mating and in distended uterine glands 15 days after mating respectively. It is concluded that the anomalous litters were derived from the fertilization of eggs ovulated at the post-partum oestrus by spermatozoa which had been retained in the female tract for at least 23 days.     

An immunological determination of specific activities of enzymes: its use in quantification of cross reactivity between enzymes of different origins.

An immunological method is presented which enables the determination of the specific activity of a pure enzyme without its extensive purification. The method consists essentially in the specific fixation of immunologically related enzymes to an immunoadsorbent containing specific antibodies raised against the wild-type form of the enzyme. We applied this method to determine the specific activity of plasmid-coded beta-galactosidases and to quantify the extent of cross-reaction between these enzymes and Escherichia coli beta-galactosidase.     

A mathematical view on the decoupled sites representation

The decoupled sites representation (DSR) is a theoretical instrument which allows to regard complex pH titration curves of biomolecules with several interacting proton binding sites as composition of isolated, non-interacting sites, each with a standard Henderson–Hasselbalch titration curve. In this work, we present the mathematical framework in which the DSR is embedded and give mathematical proofs for several statements in the periphery of the DSR. These proofs also identify exceptions. To apply the DSR to any molecule, it is necessary to extend the set of binding energies from ${\mathbb{R}}$ to a stripe within ${\mathbb{C}}$ . An important observation in this context is that even positive interaction energies (repulsion) between the binding sites will not guarantee real binding energies in the decoupled system, at least if the molecule has more than four proton binding sites. Moreover, we show that for a given overall titration curve it is not only possible to find a corresponding system with an interaction energy of zero but with any arbitrary fix interaction energy. This result also effects practical work as it shows that for any given titration curve, there is an infinite number of corresponding hypothetical molecules. Furthermore, this implies that—using a common definition of cooperative binding on the level of interaction energies—a meaningful measure of cooperativity between the binding sites cannot be defined solely on the basis of the overall titration. Consequently, all measures of cooperativity based on the overall binding curve do not measure the type of cooperativity commonly defined on the basis of interaction energies. Understanding the DSR mathematically provides the basis of transferring the DSR to biomolecules with different types of interacting ligands, such as protons and electrons, which play an important role within electron transport chains like in photosynthesis.