Site-specific chemical modification of interleukin-1 beta by acrylodan at cysteine 8 and lysine 103.

Acrylodan, which normally modifies cysteine residues, was employed to derivatize recombinant interleukin-1 beta (rIL-1 beta) under native conditions, using a reagent:protein ratio of 3:1. Two major covalent protein/acrylodan adducts were generated and subsequently purified by DEAE TSK 5PW ion exchange chromatography. Peptide mapping and mass spectrometry were used to locate the probe on the modified proteins. Both modified proteins carried one molecule of acrylodan each, one at Cys-8 and the other at Lys-103. Neither Cys-71 nor any of the other 13 lysine residues of rIL-1 beta was modified. Cysteine 71 is inaccessible to acrylodan, but the unusual specificity for Lys-103 could be caused by the location of that residue at the bottom of a hydrophobic pocket which might specifically bind the reagent. No double-labeled protein was detected, indicating that the introduction of the label at either site interferes with the labeling at the other. Both acrylodan-modified proteins exhibited bioactivity in the thymocyte proliferation assay at a level equivalent to that of the unmodified control protein (1.7 x 10(7) units/mg), which shows that the modification of either the Cys-8 or Lys-103 position with acrylodan does not interfere with the cellular bioactivities of the respective proteins. Furthermore, receptor binding assays yielded a Kd = 32.0 +/- 4.8 pM for the Lys-103-labeled protein, Kd = 69.5 +/- 12.7 pM for the unmodified protein, and Kd = 75.0 +/- 11.6 pM for the Cys-8-labeled protein. Thus, Cys-8 or Lys-103 modification of rIL-1 beta by acrylodan also does not interfere with the ability of the molecule to bind to its receptor. The slightly higher affinity of the Lys-103-labeled protein for the receptor suggests that the positive charge on this residue in the native molecule may interfere with IL-1 receptor binding. The two fluorescent labeled IL-1 proteins described herein should provide interesting probes for the study of IL-1/IL-1 receptor interactions.     

Crystal structure of type II peptide deformylase from Staphylococcus aureus

The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to, the well characterized E. coli enzyme. The most striking deviation of the S. aureus from the E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors.     

Proton,carbon, and nitrogen chemical shifts accurately delineate differences and similarities in secondary structure between the homologous proteins IRAP and IL-1β

Summary ¹H,¹³C, and¹⁵N secondary chemical shifts, defined as the difference between the observed value and the random coil value, have been calculated for interleukin-1 receptor antagonist protein and interleukin-1. Averaging of the secondary chemical shifts with those of adjacent residues was used to smooth out local effects and to obtain a correlation dependent on secondary structure. Differences and similarities in the placement of secondary structure elements in the primary segdences of these structurally homologous proteins are manifested in the smoothed secondary chemical shifts of all three types of nuclei. The close correlation observed between the secondary chemical shifts and the previously defined locations of secondary structure, as defined by traditional methods, exemplifies the advantage of chemical shifts to delineate regions of secondary structure.     

Interpretation of Changes in Circulating Tumor Cell Counts

The presence of circulating tumor cells (CTCs) in the blood of cancer patients may guide the use of therapy. We investigated how to evaluate a reduction in the number of CTCs after administration of therapy. CTCs were enumerated with the CellSearch system in 111 metastatic breast and 185 metastatic prostate cancer patients before start of a new line of chemotherapy and after initiation of therapy. Different means to express changes in CTC counts were evaluated with respect to overall survival (OS). A static CTC cutoff is the best method to determine whether a therapy is effective. This is exemplified by the highest Cox hazard ratio of 2.1 for OS; three methods to express relative differences performed worse. A lookup table is provided from which the significance of a change in CTCs can be derived. The aim of therapy should be the elimination of all CTCs. A period of 10 to 12 weeks of therapy is needed to reach the treatment effect on CTCs.     

How sympatric is speciation in the Howea palms of Lord Howe Island?

The two species of the palm genus Howea (Arecaceae) from Lord Howe Island, a minute volcanic island in the Tasman Sea, are now regarded as one of the most compelling examples of sympatric speciation, although this view is still disputed by some authors. Population genetic and ecological data are necessary to provide a more coherent and comprehensive understanding of this emerging model system. Here, we analyse data on abundance, juvenile recruitment, pollination mode and genetic variation and structure in both species. We find that Howea forsteriana is less abundant than Howea belmoreana . The genetic data based on amplified fragment length polymorphisms markers indicate similar levels of variation in the two species, despite the estimated census population size of H. belmoreana being three times larger than that of H. forsteriana . Genetic structure within species is low although some weak isolation by distance is detectable. Gene flow between species appears to be extremely limited and restricted to early-generation hybrids – only three admixed individuals, classified as F2s or first generation backcrosses to a parental species, were found among sampled palms. We conclude that speciation in Howea was indeed sympatric, although under certain strict definitions it may be called parapatric.     

Pathologies of non‐marine bivalve shells from the Late Triassic of Poland

Gorzelak, P., Nied?wiedzki, G. & Skawina, A. 2010: Pathologies of non‐marine bivalve shells from the Late Triassic of Poland. Lethaia, Vol. 43, pp. 285–289. Shells of Late Triassic non‐marine bivalves from Lisowice (Lipie ?l?skie clay pit, southern Poland), which co‐occur with remains of several vertebrate taxa (mammal‐like reptiles, carnivorous dinosaurs, pterosaurs, temnospondyl amphibians, hybodont sharks, dipnoan and ganoid fish), bear evidence of pathologies. Distribution, dimension and shape of some of these injuries (radiate tooth marks) closely match the dental morphology of lungfish (here probably represented by the genus Ceratodus). Thus, we interpret these pathologies as evidence of unsuccessful predatory attack on bivalves by this fish. This interpretation is also consistent with modern examples of such behaviour among lungfish. The feasibility that other culprits caused other pathologies (shell scarring and wedges) on the bivalves analysed is also discussed. Discovery of these traces constitutes important evidence of predator–prey interaction, which provides ‘fingerprints’ of trophic structure within this Late Triassic freshwater ecosystem. □Freshwater bivalves, lungfish, pathologies, predation, Triassic.     

High levels of genetic diversity in populations of Iris aphylla L. (Iridaceae), an endangered species in Poland

We used RAPDs (Random Amplified Polymorphic DNAs) to investigate genetic diversity and its partition within and between three populations of Iris aphylla in Poland. Analysis of Molecular Variance (AMOVA) of 84 distinct RAPD multiband genotypes revealed higher variation within populations (77.2%) than genetic differentiation between them (22.8%, P  < 0.002). Values of genetic diversity indices ( H ) were similar in all three sites (0.21–0.24). The differentiation of the populations corresponded to low average gene flow ( Nm  = 0.81). Our results indicated that genetic diversity was independent of population size. We concluded that although sexual reproduction and gene flow between populations of I. aphylla were very limited, they preserved high levels of genetic diversity. Relatively large number of seeds, which migrated in the past to populations, as well as patterns of reproduction and life history of I. aphylla may explain this situation.  © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 142 , 65–72.     

Chilled Galleria mellonella larvae: mechanism of supernumerary moulting

ABSTRACT. Supernumerary larval instars were produced when Galleria mellonella L. (Lepidoptera) larvae were chilled at 0°C. Although sensitivity to cooling stress of the last instar and younger larvae were generally the same, only penultimate and the last instar larvae showed a significant correlation between their age and the number of additional larval moults. Chilling stress induced a rapid and persistent increase in the JH titre of the last instar larvae. Severing the ventral nerve cord resulted in a predictable loss of the ability to produce supernumerary moults in chilled last instar larvae. The data suggest that sensory input stimulates allatotropic hormone secretion by the brain of chilled larvae. The possible mechanism controlling supernumerary moulting is discussed.     

Spectral characterization of environment-sensitive adducts of interleukin-1 beta.

We have determined the fluorescence properties of two covalently attached acrylodan derivatives of recombinant human interleukin-1 beta, namely the Cys-8 and Lys-103 adducts. The emission and excitation maxima indicated the presence of two operationally distinct conformers for each probe. The iodide quenching and the kinetics of fluorescence changes associated with guanidinium chloride-induced denaturation show that each covalent adduct exists both in hydrated and dehydrated environments. Furthermore, fluorescence changes associated with the binding of the adducts to a recombinant soluble murine receptor indicated that only the conformers with the label in the hydrophobic environment are competent toward the soluble murine interleukin receptor and that the hydrated and dehydrated conformers are in a dynamic equilibrium on the time scale of the binding experiments.     

Serotonin modulates a photic response in circadian locomotor rhythmicity of adults of the blow fly, Calliphora vicina

Abstract. The present experiments were undertaken to explore a role for serotonin (5-hydroxytryptamine, 5-HT) in modulating photic signal transduction in photoreceptors of the blow fly, Calliphora vicina. Injection of p-chlorophenylalanine (pCPA) into the haemolymph appeared to reduce sensitivity to the photic effects of constant ‘bright’ light (LL hyperactivity and circadian arrhythmicity). After drug injection in bright LL, flies continued with a free-running rhythm as in constant darkness (DD) or with a lengthened period τ as in ‘dim’ LL. When 5-HT was injected into flies kept in dim LL, they became hyperactive and arrhythmic as in bright LL. This finding suggests a potential role for serotonin as mediator of circadian changes in the insect visual system including extraretinal photoreceptors.