Construction of a xylanase A variant capable of polymerization

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created.     

Characterization of pNiXa,a serpin of Xenopus laevis oocytes and embryos,and its histidine-rich,Ni(II)-binding domain

A Ni(II)-binding serpin, pNiXA, is abundant in Xenopus oocytes and embryos. Kinetic assays show that purified pNiXa strongly inhibits bovine α-chymotrypsin (K₁ = 3 mM), weakly inhibits porcine elastase (K₁ = 0.5 μM), and does not inhibit bovine trypsin. The reversible, slow-binding inhibition of α-chymotrypsin by pNiXa is unaffected by Ni(II). Ovochymase in egg exudates is inhibited by pNiXa, but to a limited extent, even at high pNiXa concentrations. An octadecapeptide that models the His-rich domain (-HRHRHEQQGHHDSAKHGH-) of pNiXa forms six-coordinate, octahedral Ni(II)-complexes when the N-terminus is acetylated, and a square-planar Ni(II)-complex when the N-terminus is unblocked. Spectroscopy reveals two distinct types of octahedral Ni(II)-coordination to the N-acetylated octadecapeptide, involving, respectively, 3–4 and 5–6 imidazole nitrogens; the octadecapeptide undergoes partial, reversible precipitation in pH-and Ni(II)-dependent fashion, suggesting an insoluble, Ni(II)-coupled (Hx)_n-dimer. Such (Hx)_n-peptide interaction is confirmed by an enzyme-linked biotin-avidin assay with N-biotin-KHRHRHE-amide and N-acetyl-KHRHRHE-resin beads, which become coupled after adding Ni(II) or Zn(II). H₂O₂ oxidation of 2′-deoxyguanosine to mutagenic 8-hydroxy-2′deoxyguanosine is enhanced by the octahedral Ni(II)-octadecapeptide complex, although the effect is more intense with the square-planar Ni(II) octadecapeptide complex. Immunoperoxidase staining of whole mounts wish pNiXa antibody shows that pNiXa is distributed throughout gastrula-stage embryos and is localized during organogenesis in the brain, eye, spinal cord, myotomes, craniofacial tissues, and other sites of Ni(II) induced anomalies. Patterns of pNiXa staining are similar in controls and Ni(II)-exposed embryos. Binding of Ni(II) to pNiXa may cause embryotoxicity by enhancing oxidative reactions that produce tissue injury and genotoxicity. Although the natural target proteinases for pNiXa inhibition have not been established, pNiXa may be an important regulator of proteolysis during embryonic development. © 1996 Wiley-Liss, Inc.     

Meridianins, a new family of protein kinase inhibitors isolated from the ascidian Aplidium meridianum

Meridianins are brominated 3-(2-aminopyrimidine)-indoles which are purified from Aplidium meridianum, an Ascidian from the South Atlantic (South Georgia Islands). We here show that meridianins inhibit various protein kinases such as cyclin-dependent kinases, glycogen synthase kinase-3, cyclic nucleotide-dependent kinases and casein kinase 1. Meridianins prevent cell proliferation and induce apoptosis, a demonstration of their ability to enter cells and to interfere with the activity of kinases important for cell division and cell death. These results suggest that meridianins constitute a promising scaffold from which more potent and selective protein kinase inhibitors could be designed.     

The C‐terminal calcium‐sensitive disordered motifs regulate isoform‐specific polymerization characteristics of calsequestrin

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C‐terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn‐motif and DEXn‐motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C‐terminal motifs possess Ca²⁺‐sensitivity and affect protein function. Sequence analysis shows that both the Dn‐ and DEXn‐motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca²⁺‐mediated folding suggesting that these disordered motifs are Ca²⁺‐sensitivity. We generated chimeric proteins by swapping the C‐terminal portions between CASQ1 and CASQ2. Our studies show that the C‐terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C‐terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca²⁺‐sensitivity IDRs located at the back‐to‐back dimer interface influence isoform‐specific Ca²⁺‐dependent polymerization properties of CASQ. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 15–22, 2015.     

Interaction of Nickel(II) with histones: in vitro binding of nickel(II) to the core histone tetramer

The absorption spectra of Ni(II) bound to the core histone tetramer, (H3-H4)2, of chicken erythrocytes in 500 mM NaCl + 100 mM phosphate (pH 7.4) were recorded. A charge transfer band was seen at 317 nm, characteristic of a bond between Ni(II) and the sulfur atom of Cys-110 of histone H3. The conditional affinity constants for Ni(II) binding at pH 7.4 for low and high Ni(II) saturation (log Kc = 4.26 +/- 0.02 and 5.26 +/- 0.11 M-1, respectively) were calculated from spectrophotometric titrations with the use of this band. The binding of Ni(II) to (H3-H4)2 is proposed to involve the Cys-110 and His-113 of different H3 molecules within the tetramer. The competition between histones and low-molecular-weight chelators for Ni(II) in the cell nucleus, histidine and glutathione, is discussed on the basis of the above results, indicating that histone H3 is very likely to bind Ni(II) dissolved intracellularly from phagocytosed particulate nickel compounds.     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

A survey of scale insects in soil samples from Europe (Hemiptera,Coccomorpha)

In the last decades, several expeditions were organized in Europe by the researchers of the Hungarian Natural History Museum to collect snails, aquatic insects and soil animals (mites, springtails, nematodes, and earthworms). In this study, scale insect (Hemiptera: Coccomorpha) specimens extracted from Hungarian Natural History Museum soil samples (2970 samples in total), all of which were collected using soil and litter sampling devices, and extracted by Berlese funnel, were examined. From these samples, 43 scale insect species (Acanthococcidae 4, Coccidae 2, Micrococcidae 1, Ortheziidae 7, Pseudococcidae 21, Putoidae 1 and Rhizoecidae 7) were found in 16 European countries. In addition, a new species belonging to the family Pseudococcidae, Brevennia larvalis Kaydan, sp. n. and a new species of Ortheziidae, Ortheziola editae Szita & Konczné Benedicty, sp. n. are described and illustrated based on the adult female stage. Revised keys to the adult females of Brevennia and Ortheziola are presented.     

Lateral Dispersal and Foraging Behavior of Entomopathogenic Nematodes in the Absence and Presence of Mobile and Non-Mobile Hosts

Entomopathogenic nematodes have been classified into cruisers (active searchers) and ambushers (sit and wait foragers). However, little is known about their dispersal and foraging behavior at population level in soil. We studied lateral dispersal of the ambush foraging Steinernema carpocapsae (ALL strain) and cruise foraging Heterorhabditis bacteriophora (GPS11 strain) from infected host cadavers in microcosms (0.05 m²) containing Wooster silt-loam soil (Oxyaquic fragiudalf) and vegetation in the presence or absence of non-mobile and mobile hosts. Results showed that the presence of a non-mobile host (Galleria mellonella larva in a wire mesh cage) enhanced H. bacteriophora dispersal for up to 24 hr compared with no-host treatment, but had no impact on S. carpocapsae dispersal. In contrast, presence of a mobile host (G. mellonella larvae) increased dispersal of S. carpocapsae compared with no host treatment, but had no effect on H. bacteriophora dispersal. Also H. bacteriophora was better at infecting non-mobile than mobile hosts released into the microcosms and S. carpocapsae was better at infecting mobile than non-mobile hosts, thus affirming the established cruiser-ambusher theory. However, results also revealed that a large proportion of infective juveniles (IJs) of both species stayed near (≤ 3.8 cm) the source cadaver (88-96% S. carpocapsae; 67–79% H. bacteriophora), and the proportion of IJs reaching the farthest distance (11.4 cm) was significantly higher for S. carpocapsae (1.4%) than H. bacteriophora (0.4%) in the presence of mobile hosts. S. carpocapsae also had higher average population displacement than H. bacteriophora in the presence of both the non-mobile (5.07 vs. 3.6 cm/day) and mobile (8.06 vs. 5.3 cm/day) hosts. We conclude that the two species differ in their dispersal and foraging behavior at the population level and this behavior is affected by both the presence and absence of hosts and by their mobility.     

Age and growth of invasive round goby <Emphasis Type="Italic">Neogobius melanostomus</Emphasis> from middle Danube

Age and growth of the invasive population of round goby Neogobius melanostomus from the Slovak stretch of River Danube was examined. The samples (n=1130) were collected soon after the invasion was recorded (2004–2005), and later, when the population was already established (2008–2010). Invasive round goby in newly-occupied areas were found to reach smaller body size (15–153 mm standard length) compared to native populations. Age from 0+ to 4+, determined from scales, was recorded in both sexes. Annulus formed in April–May, which varied with age. Growth of freshly established gobies was negative allometric, suggesting increased allocation of their sources to reproduction, which corresponds to less specialized life-history. However, positive allometric growth found in longer established individuals suggests a shift in allocation towards somatic growth, which corresponds to more specialized life-history typical for native populations. None of the three parameters predicted by the theory of alternative ontogenies and invasive potential met the expectations, though two parameters, i.e. growth rate and age at maturation remain equivocal. This can be explained by too short of a time span that has elapsed from the beginning of invasion, or by ecological disturbances that have broken up otherwise stable environment in the habitat studied.     

Optical Waveguide Lightmode Spectroscopic Techniques for Investigating Membrane-Bound Ion Channel Activities

Optical waveguide lightmode spectroscopic (OWLS) techniques were probed for monitoring ion permeation through channels incorporated into artificial lipid environment. A novel sensor set-up was developed by depositing liposomes or cell-derived membrane fragments onto hydrophilic polytetrafluoroethylene (PTFE) membrane. The fibrous material of PTFE membrane could entrap lipoid vesicles and the water-filled pores provided environment for the hydrophilic domains of lipid-embedded proteins. The sensor surface was kept clean from the lipid holder PTFE membrane by a water- and ion-permeable polyethylene terephthalate (PET) mesh. The sensor set-up was tested with egg yolk lecithin liposomes containing gramicidin ion channels and with cell-derived membrane fragments enriched in GABA-gated anion channels. The method allowed monitoring the move of Na⁺ and organic cations through gramicidin channels and detecting the Cl^–-channel functions of the (α₅β₂γ₂) GABA_A receptor in the presence or absence of GABA and the competitive GABA-blocker bicuculline.