Fas-induced changes in cdc2 and cdk2 kinase activity are not sufficient for triggering apoptosis in HUT-78 cells

Recent evidence suggested a role for the cell cycle dependent kinases cdc2 and cdk2 in apoptosis. An important mechanism by which many cell types could undergo apoptosis is through the activation of the Fas molecule on the cell membrane. To investigate whether Fas-induced cell death activated cdc2 and cdk2 kinases inappropriately, the human T lymphoma cells HUT-78, which express a high copy number of Fas, and two other previously characterized subclones of the same cell line which express mutant, cell death-deficient dominant-negative forms of Fas, were Fas-challenged and the changes in cdc2 and cdk2 kinase activity monitored. In both wild-type and Fas-mutated HUT-78 cells, apoptosis was associated simultaneously with decreased cdc2 and increased cdk2 activity. This association suggested that changes in cdc2 and cdk2 kinase activity are secondary events in cell death mediated by Fas. J. Cell. Biochem. 64:579–585. © 1997 Wiley-Liss, Inc.     

Secondary metabolites released into the rhizosphere by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> spp. as underestimated component of nonspecific replant disease

Background and aims

A study was performed to investigate the role of fungal metabolites released into the rhizosphere of replanted orchards as a potential biotic component of tree growth decline.

Methods

The phytotoxicity of the gamma ray-sterilized crude culture filtrates of sixteen fungal species originating from replanted apple orchards was tested in a bioassay. Low molecular weight compounds released by Fusarium spp. were analyzed.

Results

The fungal culture filtrates affected seedling growth and health with an activity that varied from growth inhibition to promotion. Three out of the six species of Fusarium tested produced species-specific mycotoxins such as equisetin and enniatin B and D (<1 μg ml^?1 and <6 μg ml^?1, respectively) associated with root-tip necrosis, whereas fusaric acid (80–230 μg ml^?1) was associated with asymptomatic plant growth inhibition. These findings were consistent with those obtained using pure compounds. Moreover, methoxyconidiol, paecilaminol, integrastatin B and other biologically active compounds, whose fungal origin and phytotoxicity have not yet been reported, were found. in all fungal filtrates.

Conclusions

Findings suggest that i) phytopathogenicity of soil borne fungi can be expressed regardless of root infection; ii) a synergistic interaction between co-occurring mycotoxins and other biologically active compounds may explain plant growth inhibition. Iii) fungal metabolites released into soil may represent an underestimated component of nonspecific replant disease.

     

Mn(II) Oxidation Is Catalyzed by Heme Peroxidases in “Aurantimonas manganoxydans” Strain SI85-9A1 and Erythrobacter sp. Strain SD-21

A new type of manganese-oxidizing enzyme has been identified in two alphaproteobacteria, “Aurantimonas manganoxydans” strain SI85-9A1 and Erythrobacter sp. strain SD-21. These proteins were identified by tandem mass spectrometry of manganese-oxidizing bands visualized by native polyacrylamide gel electrophoresis in-gel activity assays and fast protein liquid chromatography-purified proteins. Proteins of both alphaproteobacteria contain animal heme peroxidase and hemolysin-type calcium binding domains, with the 350-kDa active Mn-oxidizing protein of A. manganoxydans containing stainable heme. The addition of both Ca²⁺ ions and H₂O₂ to the enriched protein from Aurantimonas increased manganese oxidation activity 5.9-fold, and the highest activity recorded was 700 μM min⁻¹ mg⁻¹. Mn(II) is oxidized to Mn(IV) via an Mn(III) intermediate, which is consistent with known manganese peroxidase activity in fungi. The Mn-oxidizing protein in Erythrobacter sp. strain SD-21 is 225 kDa and contains only one peroxidase domain with strong homology to the first 2,000 amino acids of the peroxidase protein from A. manganoxydans. The heme peroxidase has tentatively been named MopA (manganese-oxidizing peroxidase) and sheds new light on the molecular mechanism of Mn oxidation in prokaryotes.Mn(III,IV) oxides (Mn oxides) and soluble Mn(III) complexes are the strongest oxidizing agents in the environment after oxygen and play an important role in many biogeochemical cycles (25). At pH 7, they can oxidize metals, catalyze the formation of humic substances and organic nitrogen complexes, and oxidatively degrade humic and fulvic acids to bioavailable low-molecular-weight organic compounds (6, 38, 40). Geochemical cycling of Mn oxides can also control the distribution of many trace elements, as Mn minerals are highly charged and can adsorb and concentrate metals (22). In the pH range of aerobic natural waters (pH 6 to 8), chemical oxidation of Mn(II) is slow, but in the presence of Mn(II)-oxidizing microorganisms, the rate can be 4 to 5 orders of magnitude higher (30, 39, 48).Multicopper oxidases (MCOs) are the only identified proteins from bacteria capable of manganese oxidation. These enzymes are a class of proteins that utilize copper as a cofactor to perform four one-electron substrate oxidations, thereby reducing molecular oxygen to H₂O (37). Generally, MCOs oxidize organic compounds such as phenolics, but some fungal MCOs (laccases) that can oxidize Mn(II) to Mn(III) and Mn(IV) have been described previously (21, 36, 28). Bacterial MCOs involved with Mn oxidation have been genetically identified in Pseudomonas putida strains MnB1 and GB1 (cumA), Leptothrix discophora SS-1 (mofA), Bacillus sp. strain SG-1 (mnxG), and the alphaproteobacterium Pedomicrobium sp. strain ACM 3067 (moxA) (35). None of these MCOs share significant homology except for their copper binding motifs (35), and only in Bacillus sp. (11) have MCOs been directly linked to Mn oxidation.Another class of proteins known in eukaryotes to oxidize manganese, but not commonly identified to be involved in bacterial Mn oxidation, are heme-containing manganese peroxidases (MnPs) (5, 33). These enzymes are extremely important for the degradation activities of lignin-degrading fungi. The MnP from the basidiomycete Phanerochaete chrysosporium has a single Mn(II) binding site near the heme and produces two Mn(III) equivalents at the expense of one H₂O₂ equivalent (18, 34, 45). MnPs and MCOs are able to work in concert, with the MnP utilizing H₂O₂ produced by the MCO-catalyzed Mn(II) oxidation (36). Both types of protein produce Mn(III). While MnPs are best known to occur in fungi, a similar mechanism has been reported for a catalase/peroxidase from Mycobacterium (27), and a catalase-peroxide mechanism was suggested to be involved in Fe and Mn oxidation in Arthrobacter (13).A search for MCO-like genes in the draft genome sequence of the Mn(II)-oxidizing marine alphaproteobacterium “Aurantimonas manganoxydans” strain SI85-9A1 revealed duplicate copies of moxA-like genes (12). Initial attempts to isolate the Mn oxidase enzyme focused on the secreted proteins. Two regions were identified by a native polyacrylamide gel electrophoresis (PAGE) in-gel activity assay to have active Mn(II) oxidation, one at >250 kDa and the other at approximately 50 kDa. The active areas were excised from the gel, digested with trypsin, and analyzed with tandem mass spectrometry (MS/MS) but no peptides could be identified (12). In the same study, five bands from a corresponding Coomassie-stained sodium dodecyl sulfate (SDS)-PAGE gel returned identifications of peptides from a putative Ca²⁺ binding heme peroxidase, but no attempt was made to connect this protein to the active bands from the in-gel activity assay because they would have migrated differently (Coomassie bands were denatured protein, the in-gel activity assay native proteins).Based on the genome data, the expected size of the MoxA-like proteins was approximately 50 kDa, similar to the size of MoxA from Pedomicrobium sp. strain ACM 3067 and similar in size to the most active region from the in-gel activity assay. It was then inferred that the Mn oxidase from A. manganoxydans strain SI85-9A1 could be a Mox ortholog with an estimated size of 50 kDa that may be part of a larger >250-kDa holoenzyme (12). The experimentally identified Ca²⁺ binding heme peroxidase was suggested to be involved with the biodegradation of complex organics utilizing H₂O₂ abiotically generated by the Mn(III) produced by the Mn(II)-oxidizing MoxA-like protein (after the mechanism described by Schlosser and Höfer [36]) (12).In Erythrobacter sp. strain SD-21, the Mn oxidase enzyme was found in both the soluble and excreted fractions, suggesting that the activity may be loosely associated with the cell surface (23). Protein chromatography was employed to identify the Mn oxidase in this organism but did not conclusively implicate an MCO. The enzyme was partially purified and was found to be associated with a quinone cofactor, PQQ, that stimulated manganese oxidation in partially pure protein extracts and rescued the manganese oxidation activity in a mutant strain of Pseudomonas putida MnB1. Mn oxidation was not stimulated in vitro when copper was added, and activity was vastly decreased in the presence of MCO and quinone inhibitors. The addition of o-phenanthroline, a copper chelator and potent inhibitor of Mn oxidation in P. putida GB-1 (32), inhibited Mn oxidation only partially at concentrations far in excess of those required for GB-1. The absorbance spectrum of the partially purified protein extract did not show characteristics of an MCO, and the activity of the cell extract was between 7- and 30-fold higher than the activity measured for Mn-oxidizing organisms containing MCOs. Although the evidence pointed away from MCO involvement, H₂O₂ did not stimulate activity as expected if the enzyme was an MnP (23).A. manganoxydans strain SI85-9A1 is not easily amenable to genetic techniques, and thus, isolation of the manganese oxidase was performed through protein chromatography techniques. Since early studies failed to conclusively identify the Mn oxidase, it was decided to fractionate the proteins in the organism to localize the activity and purify the protein from the active fraction. In this work, we report the significant purification of the Mn-oxidizing protein leading to its identification as an animal heme peroxidase with multiple calcium binding motifs, and localization of the protein to the outer membrane as a peripheral membrane protein. We revisit the Mn oxidase from Erythrobacter sp. strain SD-21 (also genetically recalcitrant) and identify the protein from an active Mn-oxidizing band with a native PAGE in-gel activity assay. This protein is also an animal heme peroxidase with calcium binding motifs.     

Tissue transglutaminase in celiac disease: role of autoantibodies

In celiac disease (CD), gluten, the disease-inducing toxic component in wheat, induces the secretion of IgA-class autoantibodies which target tissue transglutaminase (tTG). These autoantibodies are produced in the small-intestinal mucosa, and, during gluten consumption, they can also be detected in patients' serum but disappear slowly from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The finding of IgA deposits on extracellular tTG in the liver, kidney, lymph nodes and muscles of patients with CD indicates that tTG is accessible to the gut-derived autoantibodies. Although the specific autoantibody response directed against tTG is very characteristic in celiac patients, their role in the immunopathology of the celiac mucosal lesion is a matter of debate. Here we report a brief summary of anti-tTG antibody effects demonstrating that these antibodies are functional and not mere bystanders in the disease pathogenesis. In fact, they inhibit intestinal epithelial cell differentiation, induce intestinal epithelial cell proliferation, increase epithelial permeability and activate monocytes and disturb angiogenesis.     

Fungal community diversity and soil health in intensive potato cropping systems of the east Po valley, northern Italy

An ecological approach was used to investigate the relationship between diversity of soil fungal communities and soil‐borne pathogen inoculum in a potato growing area of northern Italy affected by yield decline. The study was performed in 14 sites with the same tillage management practices: 10 named ‘potato sites’, that for many years had been intensely cultivated with potatoes, and 4 named ‘rotation sites’, subject to a 4‐year rotation without potatoes or any recurrent crop for many years. Fungal communities were recorded using conventional (soil fungi by plate count and endophytic fungi as infection frequency on pot‐grown potato plant roots in soil samples) and molecular approaches [Basidiomycetes and Ascomycetes with specific and denaturing gradient gel electrophoresis (DGGE) analysis]. Diversity of fungal communities in potato sites was significantly lower than that in rotation sites. In addition, fungal communities in rotation sites showed lower Berger–Parker dominance than those in the potato sites, suggesting that rotation sites had a higher diversity as well as a better fungal community balance than potato sites. The ANalysis Of SIMilarity test of soil fungi and root endophytic fungi revealed that the two cropping systems differed significantly for species composition. Root endophytic fungal communities showed a greater ability to colonise potato roots in soil samples from potato sites than those from rotation sites. Moreover, the majority of endophytic root fungal community species in potato sites belonged to the potato root rot complex and storage disease (Colletotrichum coccodes, Fusarium solani and Fusarium oxysporum), while those in rotation sites were mainly ubiquitous or saprobic fungi. Soil rDNA analyses showed that Ascomycetes were much more frequent than Basidiomycetes in all the soils examined. DGGE analysis, with the Ascomycete‐specific primer (ITS1F/ITS4A), did not reveal distinctions between the communities found at the potato and rotation sites, although the same analysis showed differences between the communities of Basidiomycetes (specific primer ITS1F/ITS4B). These findings showed that recurrent potato cropping affected diversity and composition of soil fungal communities and induced a shift in specialisation of the endophytic fungi towards potato.     

Release of free and conjugated forms of the putative pheromonal steroid 11-oxo-etiocholanolone by reproductively mature male round goby (Neogobius melanostomus Pallas, 1814)

Previous studies of the round goby (Neogobius melanostomus Pallas, 1814), an invasive fish species in the Laurentian Great Lakes of North America, have shown that this species has the ability to both synthesize and smell steroids that have a 5 beta-reduced and 3 alpha-hydroxyl (5 beta,3 alpha) configuration. An enzyme-linked immunoassay (EIA) for 3 alpha-hydroxy-5 beta-androstane-11,17-dione (11-O-ETIO) has been used to show a substantial rise in the rate of release of immunoreactive compounds into the water when males are injected with salmon gonadotropin releasing hormone analogue. Similar increases were noted for 11-ketotestosterone and 17,20 beta-dihydroxypregn-4-en-3-one. Partitioning of the extracts between diethyl ether and water showed the presence of both free and conjugated immunoreactive 11-O-ETIO. Only conjugated immunoreactivity was found in urine (implying that free steroid is released via the gills). The identity of the conjugates was probed by using HPLC, EIA, and mass spectrometry and removal of sulfate and glucosiduronate groups. Immunoreactivity in the conjugated fraction was found to be due mainly to 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-sulfate. However, the evidence was also strong for the presence in water extracts of substantial amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-glucosiduronate (which could be detected only by EIA after removal of the glucosiduronate group with beta-glucuronidase). There were also small amounts of 3 alpha-hydroxy-5 beta-androstane-11,17-dione 3-sulfate and 3 alpha,17beta-dihydroxy-5 beta-androstan-11-one 17-glucosiduronate. These studies give some idea of the types, amounts, and ratios of 11-O-ETIO derivatives that are released by reproductive N. melanostomus and will aid further research into the putative pheromonal roles of 5 beta,3 alpha-reduced androgens in this species.     

Strong conservation of the bird Z chromosome in reptilian genomes is revealed by comparative painting despite 275 million years divergence

The divergence of lineages leading to extant squamate reptiles (lizards, snakes, and amphisbaenians) and birds occurred about 275 million years ago. Birds, unlike squamates, have karyotypes that are typified by the presence of a number of very small chromosomes. Hence, a number of chromosome rearrangements might be expected between bird and squamate genomes. We used chromosome-specific DNA from flow-sorted chicken (Gallus gallus) Z sex chromosomes as a probe in cross-species hybridization to metaphase spreads of 28 species from 17 families representing most main squamate lineages and single species of crocodiles and turtles. In all but one case, the Z chromosome was conserved intact despite very ancient divergence of sauropsid lineages. Furthermore, the probe painted an autosomal region in seven species from our sample with characterized sex chromosomes, and this provides evidence against an ancestral avian-like system of sex determination in Squamata. The avian Z chromosome synteny is, therefore, conserved albeit it is not a sex chromosome in these squamate species.