Evidence for proteins involved in prophenoloxidase cascade Eisenia fetida earthworms

The prophenoloxidase cascade represents one of the most important defense mechanisms in many invertebrates. Following the recognition of microbial saccharides by pattern recognition molecules, proteinases cleave inactive prophenoloxidase to its active form, phenoloxidase. Phenoloxidase is a key enzyme responsible for the catalysis of the melanization reaction. Final product melanin is involved in wound healing and immune responses. Prophenoloxidase cascade has been widely described in arthropods; data in other invertebrate groups are less frequent. Here we show detectable phenoloxidase activity in 90-kDa fraction of the coelomic fluid of earthworms Eisenia fetida. Amino acid sequencing of peptides from the active fraction revealed a partial homology with invertebrate phenoloxidases and hemocyanins. Moreover, the level of phenoloxidase activity is lower and the activation slower as compared to other invertebrates.     

Oncostatin M up-regulates low density lipoprotein receptors in HepG2 cells by a novel mechanism.

Oncostatin M is a growth regulatory protein secreted by macrophages and activated T lymphocytes. In a hepatoma cell line (HepG2) the polypeptide very potently increased low density lipoprotein (LDL) uptake with an EC50 of 0.1-0.2 nM. The stimulation of LDL uptake was detectable by 2 h, was maximal by 8 h, and remained elevated through 20 h of oncostatin M incubation. In a similar fashion, oncostatin M also increased the number of cell surface LDL receptors by a mechanism that was inhibited by cycloheximide or the protein kinase C inhibitor H-7. Oncostatin M stimulation of LDL uptake and receptor protein occurred regardless of the state of cholesterol-dependent regulation of HepG2 LDL receptor (i.e. cells incubated in medium containing lipoproteins responded to the same extent as did cells incubated in the absence of lipoproteins). No significant effects were observed on sterol synthesis over 8 h or on DNA synthesis over 24 h. Oncostatin M induced rapid alterations in HepG2 phospholipid metabolism. Within 5-15 min there was a 20-50% increase in incorporation of 32P into several classes of phospholipids, including the phosphoinositides. Radiolabeled diacylglycerol levels were elevated 20% by 2 min and nearly 50% by 15 min. In addition, the polypeptide induced rapid increased (within 1 min) in phosphorylation of HepG proteins on tyrosine residues. Stimulation of both phosphotyrosine and LDL receptor up-regulation by oncostatin M was decreased by the tyrosine kinase inhibitor genistein. We propose that oncostatin M up-regulates HepG2 LDL receptor expression by a mechanism that includes stimulation of a tyrosine kinase followed by generation of phospholipid-related second messengers.     

Zinc(II) chlorido complexes of protonated kinetin and its derivatives: Synthesis, properties and X-ray structure of [Zn(Hkinetin)Cl3]·kinetin

The syntheses and characterization of five novel zinc(II) complexes with protonated kinetin (6-furfurylaminopurine) and its derivatives are described. Based on the results following from elemental analyses (C, H, N), FTIR, Raman, ¹H and ¹³C NMR spectroscopy, conductivity measurements, thermogravimetric (TG) and differential thermal analyses (DTA), and single crystal X-ray analysis, the complexes of the general composition [Zn(HLⁿ)Cl₃]·xLⁿ (1-5) have been prepared, where L¹ = kinetin (6-furfurylaminopurine), L² = 6-(5-methylfurfurylamino)purine, L³ = 2-chloro-6-furfurylaminopurine, L⁴ = 2-chloro-6-(5-methylfurfurylamino)purine and L⁵ = 2-chloro-6-furfurylamino-9-isopropylpurine, and x = 1/2-2. The structure of [Zn(HL¹)Cl₃]·L¹ (1) has been determined by single crystal X-ray analysis. The Zn(II) atom is tetrahedrally coordinated by three chlorido ligands and one N3-protonated organic molecule forming a ZnCl₃N donor set. The organic ligand L¹ is coordinated to the Zn(II) centre through the N7 atom of the purine moiety. NMR spectroscopic study confirmed the N3 and N7 atom to be the protonation, and coordination site, respectively.     

Fluorescence spectroscopy studies of HEK293 cells expressing DOR-Gi1α fusion protein; the effect of cholesterol depletion

Biophysical studies of fluorescence anisotropy of DPH and Laurdan generalized polarization were performed in plasma membranes (PM) isolated from control and cholesterol-depleted HEK293 cells stably expressing pertussis toxin (PTX)-insensitive DOR-Gi1α (Cys351-Ile351) fusion protein. PM isolated from control, PTX-untreated, cells were compared with PM isolated from PTX-treated cells. Results from both types of PM indicated that i) hydrophobic membrane interior was made more accessible to water molecules and more chaotically organized in cholesterol-depleted samples, ii) cholesterol depletion resulted in an overall increase in surface area of membrane, membrane fluidity, and mobility of its constituents. Analysis of DOR-Gi1α coupling in PTX-treated and PTX-untreated cells indicated that cholesterol depletion did not alter the agonist binding site of DOR (Bmax and Kd) but the ability of DOR agonist DADLE to activate G proteins was markedly impaired. In PTX-untreated membranes, EC50 for DADLE-stimulated [35S]GTPγS binding was shifted by one order of magnitude to the right: from 4.3±1.2×10(-9) M to 2.2±1.3×10(-8) M in control and cholesterol-depleted membrane samples, respectively. In PTX-treated membranes, EC50 was shifted from 4.5±1.1×10(-9) M to 2.8±1.4×10(-8) M. In summary, the perturbation of optimum PM organization by cholesterol depletion deteriorates functional coupling of DOR to covalently bound Gi1α as well as endogenously expressed PTX-sensitive G proteins of Gi/Go family while receptor ligand binding site is unchanged. The biophysical state of hydrophobic plasma (cell) membrane interior should be regarded as regulatory factor of DOR-signaling cascade.     

Arbuscular mycorrhizal symbiosis on serpentine soils: the effect of native fungal communities on different Knautia arvensis ecotypes

Serpentine soils represent a unique environment that imposes multiple stresses on vegetation (low Ca/Mg ratios, macronutrient deficiencies, elevated heavy metal concentrations and drought). Under these conditions, a substantial role of arbuscular mycorrhizal (AM) symbiosis can be anticipated due to its importance for plant nutrition and stress alleviation. We tested whether serpentine and non-serpentine populations of Knautia arvensis (Dipsacaceae) differ in the benefits derived from native AM fungal communities. Four serpentine and four non-serpentine populations were characterised in terms of mycorrhizal colonisation and soil characteristics. The serpentine populations showed significantly lower mycorrhizal colonisation than their non-serpentine counterparts. The mycorrhizal colonisation positively correlated with soil pH, Ca and K concentrations and Ca/Mg ratio. Seedlings from each population were then grown for 3 months in their sterilised native substrates, either uninoculated or reinoculated with native AM fungi. Two serpentine and two non-serpentine populations responded positively to mycorrhizal inoculation, while no significant change in plant growth was observed in the remaining populations. Contrary to our hypothesis, serpentine populations of K. arvensis did not show higher mycorrhizal growth dependence than non-serpentine populations when grown in their native soils and inoculated with native AM fungi.     

Mycobacterial screening of Czech red deer (Cervus elaphus) populations in overwintering sites, 2004-2006

One isolate of Mycobacterium avium subsp. paratuberculosis was detected in 2,212 fecal samples of wild deer assembled in overwintering sites (OwS). Neither M. bovis nor M. a. subsp. avium was found. Therefore, congregating deer in OwSs does not automatically lead to the amplification of these pathogens among animals in OWSs.     

Expressivity of apomixis in 2n?+?n hybrids from an apomictic and a sexual parent: insights into variation detected in Pilosella (Asteraceae: Lactuceae)

Reproductive variation was studied in the tetraploid Pilosella aurantiaca, hexaploid P. rubra (both species with facultative autonomous apospory) and in their 2n + n hybrids, which were obtained by crossing with a sexual pollen parent (tetraploid P. officinarum). The different DNA content in P. aurantiaca and P. officinarum demonstrated the actual 2n + n origin, both spontaneous from the field and through experimental crosses, of their hexaploid hybrids. The octoploid 2n + n progeny were recovered from an experimental cross of P. rubra and P. officinarum. The reproductive pathways operating in two maternal facultatively apomictic species and in the hybrids were quantified using a flow cytometric analysis of seeds obtained from either open-pollinated or emasculated plants. Whereas both maternal species displayed a high penetrance of apomixis, the level of apomixis among the majority of 2n + n hybrids was much lower and variable. Some of the hexaploid hybrids had a reduced seed set. Compared to the respective maternal parents, the decrease in apomixis due to haploid parthenogenesis and/or n + n mating was evident in almost all unreduced hybrids, irrespective of their field/experimental origin and ploidy. Hence, the reproductive behaviour in the apomictic maternal parent was profoundly different from that of the 2n + n hybrids with a sexual parent in spite of the preservation of the complete maternal genome in the hybrids. The regulatory interactions in hybrid genomes, such as effects of modifiers, heterochrony, and epigenetic control, may be consistent with the different expressivity of apomixis observed under different genetic backgrounds.