Autoactivation of human ADAM8: a novel pre-processing step is required for catalytic activity

Members of the ADAM (a disintegrin and metalloproteinase) family of proteins possess a multidomain architecture which permits functionalities as adhesion molecules, signalling intermediates and proteolytic enzymes. ADAM8 is found on immune cells and is induced by multiple pro-inflammatory stimuli suggesting a role in inflammation. Here we describe an activation mechanism for recombinant human ADAM8 that is independent from classical PC (pro-protein convertase)-mediated activation. N-terminal sequencing revealed that, unlike other ADAMs, ADAM8 undergoes pre-processing at Glu(158), which fractures the Pro (pro-segment)-domain before terminal activation takes place to remove the putative cysteine switch (Cys(167)). ADAM8 lacking the DIS (disintegrin) and/or CR (cysteine-rich) and EGF (epidermal growth factor) domains displayed impaired ability to complete this event. Thus pre-processing of the Pro-domain is co-ordinated by DIS and CR/EGF domains. Furthermore, by placing an EK (enterokinase) recognition motif between the Pro- and catalytic domains of multiple constructs, we were able to artificially remove the pro-segment prior to pre-processing. In the absence of pre-processing of the Pro-domain a marked decrease in specific activity was observed with the autoactivated enzyme, suggesting that the Pro-domain continued to associate and inhibit active enzyme. Thus, pre-processing of the Pro-domain of human ADAM8 is important for enzyme maturation by preventing re-association of the pro-segment with the catalytic domain. Given the observed necessity of DIS and CR/EGF for pre-processing, we conclude that these domains are crucial for the proper activation and maturation of human ADAM8.     

The Upper Cretaceous belemnite <Emphasis Type="Italic">Praeactinocamax plenus</Emphasis> (Blainville, 1827) from Lower Saxony (Upper Cenomanian,northwest Germany) and its distribution pattern in Europe

Occurrences of the Upper Cenomanian (Upper Cretaceous) belemnite Praeactinocamax plenus from the plenus Bed of northwest Germany (Söhlde-Loges working quarry near Salzgitter, Lower Saxony) are documented and described for the first time on the basis of two in situ finds. The find horizon and its surrounding beds are re-evaluated in a sequence stratigraphical context. In contrast to the interpretations of other authors, the plenus Bed is seen as a pelagization event in a parasequence of transgressively stacked beds, delimited by two significant erosion surfaces below and above. The exclusive occurrence of P. plenus in the top part of the plenus Bed and its absence from the post-plenus Bed succession, in the equivalent of which (higher part of the Plenus Marls Member) it is very common in southern England (Anglo-Paris Basin), is explained by ecological factors in stratigraphically complete sections (intra-shelf depressions) and by gaps in the stratigraphic records in swell settings. The distribution pattern of P. plenus suggests a preference for nearshore settings and a demersal mode of life.     

Synthesis of spirocyclic σ1 receptor ligands as potential PET radiotracers,structure–affinity relationships and in vitro metabolic stability

Several 3H-spiro[[2]benzofuran-1,4′-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF₃·OEt₂ catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure–affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar σ₁ affinity (K_i = 0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the σ₂ subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [¹⁸F]2a and [¹⁸F]2e two different radiosynthetic approaches were followed.     

Micropropagation by tissue culture triggers differential expression of infectious endogenous Banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus ) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Isolation and characterisation of bacteria from the Eastern Mediterranean deep sea

The Eastern Mediterranean deep sea is one of the most oligotrophic regions in the world’s ocean. With the aim to classify bacteria from this special environment we isolated 107 strains affiliating to the Gammaproteobacteria, Alphaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes from sediments of the Eastern Mediterranean Sea. As determined by 16S rRNA gene sequence analysis, Actinobacteria and Firmicutes, in particular members of the genus Bacillus, were dominant and represented a remarkable diversity with 27 out of a total of 33 operational taxonomic units obtained from the untreated sediment. The considerable percentage of operational taxonomic units (42%) which may be considered to be new species underlines the uniqueness of the studied environment. In order to selectively enrich bacteria which are adapted to the deep-sea conditions and tolerate broad pressure ranges, enrichments were set up with a sediment sample under in situ pressure and temperature (28 MPa, 13.5°C) using N-acetyl-d-glucosamine as substrate. Interestingly Gammaproteobacteria were significantly enriched and dominant among the strains isolated after pressure pre-incubation. Obviously, Gammaproteobacteria have a selective advantage under the enrichment conditions applied mimicking nutrient supply under pressure conditions and cope well with sudden changes of hydrostatic pressure. However, under the continued low nutrient situation in the Eastern Mediterranean deep-sea sediments apparently Firmicutes and Actinobacteria have a clear adaptative advantage.     

Tissue distribution of L-fucokinase in rodents

L-fucose (fucose) is a monosaccharide normally present in mammals and is unique in being the only levorotatory sugar that can be synthesized and utilized by mammals. The metabolism of fucose is incompletely understood, but fucose can be synthesized de novo or salvaged. The utilization of fucose in the salvage pathway begins with phosphorylation by fucokinase. As part of an investigation of fucose metabolism in normal and disease states, we began an investigation of this enzyme. In this report, we present the tissue distribution of the enzyme in rat and mouse. The highest amount of activity was present in brain of both species. Some activity was found in all tissues examined (liver, kidney, heart, lung, spleen, brain, muscle, thymus, white adipose, testes, eye, aorta, small intestine, and submaxillary gland). Very low levels were found in small intestine. Varying levels in the tissues seems most likely to be the result of varying amounts of fucokinase protein as no difference in the Km values of crude enzyme could be shown. Protein-bound fucose levels were determined using the L-cysteine-phenol-sulfuric acid (CPS) assay. There is not a good correlation between fucokinase activity and protein-bound fucose, suggesting some tissues are more active in synthesis of fucose than others.     

Cloning of a Picea abies monosaccharide transporter gene and expression – analysis in plant tissues and ectomycorrhizas

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H⁺/monosaccharide transporters from Ara- bidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas. Received: 9 October 1999 / Accepted: 22 December 1999     

Resibufogenin corrects hypertension in a rat model of human preeclampsia

The study of the pathogenesis of preeclampsia has been hampered by a relative dearth of animal models. We developed a rat model of preeclampsia in which the excretion of a circulating inhibitor of Na/K ATPase, marinobufagenin (MBG), is elevated. These animals develop hypertension, proteinuria, and intrauterine growth restriction. The administration of a congener of MBG, resibufogenin (RBG), reduces blood pressure to normal in these animals, as is the case when given to pregnant animals rendered hypertensive by the administration of MBG. Studies of Na/K ATPase inhibition by MBG and RBG reveal that these agents are equally effective as inhibitors of the enzyme.     

Effects of moderate ammonium enrichment on three submersed macrophytes under contrasting light availability

1. Increased ammonium concentrations and decreased light availability in a water column have been reported to adversely affect submersed vegetation in eutrophic waters worldwide. 2. We studied the chronic effects of moderate enrichment (NH₄–N: 0.16–0.25 mg L^?1) on the growth and carbon and nitrogen metabolism of three macrophytes (Ceratophyllum demersum, Myriophyllum spicatum and Vallisneria natans) under contrasting light availability in a 2‐month experiment. 3. The enrichment greatly increased the contents of free amino acids and nitrogen in the shoot / leaf of the macrophytes. This indicates that was the dominant N source for the macrophytes. 4. Soluble carbohydrate contents remained relatively stable in the shoot / leaf of the macrophytes irrespective of the treatments. Under ambient light, the starch contents in the shoot / leaf of C. demersum and M. spicatum increased with enrichment, whereas V. natans did not exhibit any change. The starch contents decreased in C. demersum, increased in M. spicatum and remained unchanged in V. natans after the combined treatment of enrichment and reduced light. 5. The enrichment did not affect the growth of the three macrophytes under the ambient light. However, it did suppress the growth of C. demersum and M. spicatum under the reduced light. The results indicate that a moderate enrichment was not directly toxic to the macrophytes although it might change their viability in eutrophic lakes in terms of the carbon and nitrogen metabolism.