Pseudomonas exotoxin exhibits increased sensitivity to furin when sequences at the cleavage site are mutated to resemble the arginine-rich loop of diphtheria toxin

To be toxic for mammalian cells, Pseudomonas exotoxin (PE) requires proteolytic cleavage between Arg-279 and Gly-280. Cleavage, which is mediated by the cellular protease furin, generates an active C-terminal fragment which translocates to the cytosol and inhibits protein synthesis. In vitro , furin-mediated cleavage is optimal at pH 5.5 with a relatively slow turnover rate. Within cells, only 5–10% of cell-associated PE is cleaved. To investigate the reasons for this inefficient cleavage, the amino acid composition near the cleavage site was altered to resemble more closely the arginine-rich sequence from the functionally similar region of diphtheria toxin (DT). Four PE-DT mutants were generated, whereby 1, 5, 6 or 8 amino acids at the PE-cleavage site were changed to amino acids found at the DT-cleavage site. Mutant proteins were expressed in Escherichia coli , purified and then analysed for their susceptibility to cleavage by furin and trypsin, susceptibility to cell-mediated cleavage, and cytotoxic activity relative to wild-type PE. At pH 5.5, the rate of both furin-mediated cleavage and trypsin-mediated cleavage increased dramatically when amino acids in PE were altered to resemble the DT sequence. This increase did not alter the pH optimum for furin-mediated cleavage of PE toxins, which remained at pH 5.0–5.5. When radioactive versions of selected PE-DT proteins were added to intact cells, an increase in the percentage of molecules that were cleaved relative to wild-type PE was also seen. However, changes that favoured increased proteolysis apparently interfered with other important toxin functions because none of the PE-DT proteins exhibited enhanced toxicity for cells when compared with the activity of wild-type PE.     

A family with a satellited Yq chromosome

Summary A large pedigree with a satellited Yq chromosome is described, Q, C, and NOR banding were performed. Family C proband suffers from a Klinefelter syndrome.     

Characterization and determination of titratable groups of proteins by linearization of titration curves. II. Application to lysozyme

The potentiometric acid-base titration curve of fully protonated lysozyme at ionic strengths of 0.10 and 1.0 m has been performed. The stoichiometry and the pK_a values of each titratable group have been determined through the linearization of titration curves. Two types of carboxylic groups with pK_a values of 3.76 and 5.02, the imidazole group with pK_a 7.37 and the amine group with pK_a 9.63, have been identified at an ionic strength of 0.10 m at 25.0°C. The number of titratable groups found per mole of protein has been 5.12 and 5.60 for the two types of carboxylic groups, 1.13 for the imidazole group, and 3.19 for the amino groups. The endpoint of the titration of the protein obtained by this method accords quite well with the endpoint obtained by the use of Gran function applied to the excess of strong base.     

Genetic and functional analysis of the basic replicon of pPS10, a plasmid specific for Pseudomonas isolated from Pseudomonas syringae patovar savastanoi.

The sequence of a 1823 base-pair region containing the replication functions of pPS10, a narrow host-range plasmid isolated from a strain of Pseudomonas savastanoi, is reported. The origin of replication, oriV, or pPS10 is contained in a 535 base-pair fragment of this sequence that can replicate in the presence of trans-acting function(s) of the plasmid. oriV contains four iterons of 22 base-pairs that are preceded by G+C-rich and A+T-rich regions. A dnaA box located adjacent to the repeats of the origin is dispensable but required for efficient replication of pPS10; A and T are equivalent bases at the 5' end of the box. repA, the gene of a trans-acting replication protein of 26,700 Mr has been identified by genetic and functional analysis. repA is adjacent to the origin of replication and is preceded by the consensus sequences of a typical sigma 70 promoter of Escherichia coli. The RepA protein has been identified, using the minicell system of E. coli, as a polypeptide with an apparent molecular mass of 26,000. A minimal pPS10 replicon has been defined to a continuous 1267 base-pair region of pPS10 that includes the oriV and repA sequences.     

The orphan receptor cDNA RDC4 encodes a 5-HT1D serotonin receptor.

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.     

Distribution of muscarinic receptor subtypes in rat brain as determined in binding studies with AF-DX 116 and pirenzepine

In vitro competition binding experiments with the selective muscarinic antagonists AF-DX 116 and pirenzepine (PZ) vs 3H-N-methylscopolamine as radioligand revealed a characteristic distribution of muscarinic receptor subtypes in different regions of rat brain. Based on non linear least squares analysis, the binding data were compatible with the presence of three different subtypes: the M1 receptor (high affinity for PZ), the cardiac M2 receptor (high affinity for AF-DX 116) and the glandular M2 receptor (low affinity for PZ and AF-DX 116). The highest proportion of M1 receptors was found in the hippocampus, whilst the cerebellum and the hypothalamus were the regions with the largest fraction of the cardiac M2 and glandular M2 receptors, respectively. In certain brain areas, depending on the relative proportions of the subtypes, flat binding curves were seen for AF-DX 116 and PZ. Based on these data, an approximate distribution pattern of the subtypes in the various brain regions is presented.     

Cryptic phenology in plants: Case studies,implications, and recommendations

Plant phenology—the timing of cyclic or recurrent biological events in plants—offers insight into the ecology, evolution, and seasonality of plant‐mediated ecosystem processes. Traditionally studied phenologies are readily apparent, such as flowering events, germination timing, and season‐initiating budbreak. However, a broad range of phenologies that are fundamental to the ecology and evolution of plants, and to global biogeochemical cycles and climate change predictions, have been neglected because they are “cryptic”—that is, hidden from view (e.g., root production) or difficult to distinguish and interpret based on common measurements at typical scales of examination (e.g., leaf turnover in evergreen forests). We illustrate how capturing cryptic phenology can advance scientific understanding with two case studies: wood phenology in a deciduous forest of the northeastern USA and leaf phenology in tropical evergreen forests of Amazonia. Drawing on these case studies and other literature, we argue that conceptualizing and characterizing cryptic plant phenology is needed for understanding and accurate prediction at many scales from organisms to ecosystems. We recommend avenues of empirical and modeling research to accelerate discovery of cryptic phenological patterns, to understand their causes and consequences, and to represent these processes in terrestrial biosphere models.     

The inositol phosphatase SHIP-1 inhibits NOD2-induced NF-κB activation by disturbing the interaction of XIAP with RIP2

SHIP-1 is an inositol phosphatase predominantly expressed in hematopoietic cells. Over the ten past years, SHIP-1 has been described as an important regulator of immune functions. Here, we characterize a new inhibitory function for SHIP-1 in NOD2 signaling. NOD2 is a crucial cytoplasmic bacterial sensor that activates proinflammatory and antimicrobial responses upon bacterial invasion. We observed that SHIP-1 decreases NOD2-induced NF-κB activation in macrophages. This negative regulation relies on its interaction with XIAP. Indeed, we observed that XIAP is an essential mediator of the NOD2 signaling pathway that enables proper NF-κB activation in macrophages. Upon NOD2 activation, SHIP-1 C-terminal proline rich domain (PRD) interacts with XIAP, thereby disturbing the interaction between XIAP and RIP2 in order to decrease NF-κB signaling.     

Wing coloration and pigment gradients in scales of pierid butterflies

Depending on the species, the individual scales of butterfly wings have a longitudinal gradient in structure and reflectance properties, as shown by scanning electron microscopy and microspectrophotometry. White scales of the male Small White, Pieris rapae crucivora, show a strong gradient in both the density in pigment granules and the reflectance. After pigment extraction by aqueous ammonia, scales of male P. r. crucivora closely resemble the unpigmented scales of female P. r. crucivora. Only a minor gradient exists in the white and orange scales of the male Orange Tip, Anthocharis cardamines. Pigment extraction of orange scales of A. cardamines causes bleaching. Partial bleaching transforms the scales so that they resemble certain scales of Phoebis philea that have a natural extreme gradient. Reflectance measurements on an artificial stack of two overlapping scales as well as on the scale stacks existing on intact and partially denuded wings of the Large White, Pieris brassicae, quantitatively demonstrate the reflectance enhancement by scale stacking.