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.     

Upstream of N-Ras C-terminal cold shock domains mediate poly(A) specificity in a novel RNA recognition mode and bind poly(A) binding protein

RNA binding proteins (RBPs) often engage multiple RNA binding domains (RBDs) to increase target specificity and affinity. However, the complexity of target recognition of multiple RBDs remains largely unexplored. Here we use Upstream of N-Ras (Unr), a multidomain RBP, to demonstrate how multiple RBDs orchestrate target specificity. A crystal structure of the three C-terminal RNA binding cold-shock domains (CSD) of Unr bound to a poly(A) sequence exemplifies how recognition goes beyond the classical ππ-stacking in CSDs. Further structural studies reveal several interaction surfaces between the N-terminal and C-terminal part of Unr with the poly(A)-binding protein (pAbp). All interactions are validated by mutational analyses and the high-resolution structures presented here will guide further studies to understand how both proteins act together in cellular processes.     

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.