Genetic Evidence for Pak1 Autoinhibition and Its Release by Cdc42

Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.     

The adrenocortical stress-response of Black-legged Kittiwake chicks in relation to dietary restrictions

In this study we examined hormonal responses of Black-legged Kittiwake (Rissatridactyla) chicks to experimental variations in energy content and nutritional quality (low or high lipid to protein ratio, LPR) of their food. Starting at the age of 10 days, chicks were fed either high or low LPR fish at 30, 50, 70 and 100% of ad libitum energy intake. After 20 days of treatment, chicks were exposed to a standardized acute handling and restraint stress protocol, where a baseline sample was taken immediately after taking a chick from the nest, and three additional blood samples were taken at intervals up to 50 min. Testosterone and corticosterone titres in plasma were measured via radioimmunoassay. We found that baseline testosterone levels were not significantly affected by the experimental treatments. Food-restricted chicks had elevated baseline and acute stress-induced levels of corticosterone compared to chicks fed ad libitum. An elevation of circulating levels of corticosterone in energetically stressed individuals was further magnified by low nutritional quality of food. Baseline and acute stress-induced corticosterone levels of chicks were negatively correlated with their fat reserves. We conclude that the physiological condition of Black-legged Kittiwake chicks can be assessed reliably by measuring circulating levels of corticosterone. We discuss short- and long-term effects of elevated corticosterone secretion in food-stressed nest-bound chicks. Accepted: 13 April 1999     

Quorum-sensing cross talk: isolation and chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other Gram-negative bacteria

In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively. These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(DeltaAla-L-Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro). Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.     

Genetic structure and gene flow among Komodo dragon populations inferredby microsatellite loci analysis

A general concern for the conservation of endangered species is the maintenance of genetic variation within populations, particularly when they become isolated and reduced in size. Estimates of gene flow and effective population size are therefore important for any conservation initiative directed to the long-term persistence of a species in its natural habitat. In the present study, 10 microsatellite loci were used to assess the level of genetic variability among populations of the Komodo dragon Varanus komodoensis. Effective population size was calculated and gene flow estimates were compared with palaeogeographic data in order to assess the degree of vulnerability of four island populations. Rinca and Flores, currently separated by an isthmus of about 200 m, retained a high level of genetic diversity and showed a high degree of genetic similarity, with gene flow values close to one migrant per generation. The island of Komodo showed by far the highest levels of genetic divergence, and its allelic distinctiveness was considered of great importance in the maintenance of genetic variability within the species. A lack of distinct alleles and low levels of gene flow and genetic variability were found for the small population of Gili Motang island, which was identified as vulnerable to stochastic threats. Our results are potentially important for both the short- and long-term management of the Komodo dragon, and are critical in view of future re-introduction or augmentation in areas where the species is now extinct or depleted.     

CeLINC,a fluorescence-based protein–protein interaction assay in Caenorhabditis elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein–protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein–protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.     

Complexation of trypsin and alcohol dehydrogenase with poly(diallyldimethylammonium chloride)

Complexation of alcohol dehydrogenase (ADH) and trypsin with poly(diallyldimethyl-ammonium chloride) (PDADMAC) in dilute electrolyte solution was studied by turbidimetric titration, quasi-elastic light scattering (QELS), and electrophoretic light scattering (ELS). Both QELS and turbidimetric titration show that PDADMAC forms complexes with ADH and trypsin in 0.01M NaCl solution at pH ≥ 6.8 and pH ≥ 9.2, respectively. These complexes take the form of stable coacervates in 0.01M, pH 11.0, phosphate buffer solution. QELS shows sizes of 400 and 315 nm for the coacervates of ADH-PDMDAAC and trypsin-PDMDAAC, respectively, while ELS reveals that these coacervates carry a net positive charge. Activity measurements show that both ADH and trypsin are enzymatically active in their coacervated states. Complexation of trypsin and PDADMAC was also studied by fluorescence in 0.01M, pH 11.0, phosphate buffer, and the protein emission was found to be quenched by complexation. The fluorescence quenching data show that trypsin retains its three-dimensional structure in the complex. These and other results are consistent with the quenching of the two tryptophans on the protein surface, but not the interior ones.© 1997 John Wiley & Sons, Inc.