Pyrroloquinoline-quinone: a reactive oxygen species scavenger in bacteria

Transgenic Escherichia coli expressing pyrroloquinoline-quinone (PQQ) synthase gene from Deinococcus radiodurans showed superior survival during Rose Bengal induced oxidative stress. Such cells showed significantly low levels of protein carbonylation as compared to non-transgenic control. In vitro, PQQ reacted with reactive oxygen species with rate constants comparable to other well known antioxidants, producing non-reactive molecular products. PQQ also protected plasmid DNA and proteins from the oxidative damage caused by gamma-irradiation in solution. The data suggest that radioprotective/oxidative stress protective ability of PQQ in bacteria may be consequent to scavenging of reactive oxygen species per se and induction of other free radical scavenging mechanism.     

ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg(220) (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave alpha(2)-macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family.     

Discovery and characterization of a novel, widely expressed metalloprotease, ADAMTS10, and its proteolytic activation

We describe the discovery and characterization of ADAMTS10, a novel metalloprotease encoded by a locus on human chromosome 19 and mouse chromosome 17. ADAMTS10 has the typical modular organization of the ADAMTS family, with five thrombospondin type 1 repeats and a cysteine-rich PLAC (protease and lacunin) domain at the carboxyl terminus. Its domain organization and primary structure is similar to a novel long form of ADAMTS6. In contrast to many ADAMTS proteases, ADAMTS10 is widely expressed in adult tissues and throughout mouse embryo development. In situ hybridization analysis showed widespread expression of Adamts10 in the mouse embryo until 12.5 days of gestation, after which it is then expressed in a more restricted fashion, with especially strong expression in developing lung, bone, and craniofacial region. Mesenchymal, not epithelial, expression in the developing lung, kidney, gonad, salivary gland, and gastrointestinal tract is a consistent feature of Adamts10 regulation. N-terminal sequencing and treatment with decanoyl-Arg-Val-Lys-Arg-chloromethylketone indicate that the ADAMTS10 zymogen is processed by a subtilisin-like proprotein convertase at two sites (Arg64/Gly and Arg233/Ser). The widespread expression of ADAMTS10 suggests that furin, a ubiquitously expressed proprotein convertase, is the likely processing enzyme. ADAMTS10 expressed in HEK293F and COS-1 cells is N-glycosylated and is secreted into the medium, as well as sequestered at the cell surface and extracellular matrix, as demonstrated by cell surface biotinylation and immunolocalization in nonpermeabilized cells. ADAMTS10 is a functional metalloprotease as demonstrated by cleavage of alpha2-macroglobulin, although physiological substrates are presently unknown.     

Pyrroloquinoline-quinone synthesized in Escherichia coli by pyrroloquinoline-quinone synthase of Deinococcus radiodurans plays a role beyond mineral phosphate solubilization

Deinococcus radiodurans, an extremely radioresistant bacterium, synthesizes coenzyme pyrroloquinoline-quinone (PQQ) but exhibits a negative phenotype for mineral phosphate solubilization. Gene for the putative PQQ synthesizing protein was PCR amplified and cloned from Deinococcus, sequenced, and expressed in Escherichia coli, under an inducible E. coli promoter. The transgenic E. coli expressed PQQ synthase protein of 42kDa and complemented the mineral phosphate solubilization phenotype of E. coli, suggesting the synthesis of an active protein. The cells expressing high levels of this protein showed increased protection against photodynamically produced reactive oxygen species. The effect could be attributed to the upregulation of antioxidant enzymes such as catalase and superoxide dismutase by PQQ in transgenic E. coli through an unknown mechanism. The study elucidates a hitherto unknown possible function of PQQ in bacteria.     

Population genetic subdivision in the New Zealand greenshell mussel (Perna canaliculus) inferred from single-strand conformation polymorphism analysis of mitochondrial DNA

Single-strand conformation polymorphism (SSCP) analysis of the NADH IV region of the mitochondrial DNA (mtDNA) molecule in greenshell mussels (Perna canaliculus) indicated strong population genetic structuring in this endemic New Zealand species. A northern and a southern group were differentiated by frequency shifts in common haplotypes and by the occurrence of a unique southern haplotype at approximately 20% frequency. This split occurred south of Cook Strait (the body of water between the North and the South Island) at approximately 42 degrees S latitude. Northern populations were less genetically diverse than southern populations and mussels from the west coast of the South Island were most distinct from northern mussels. We hypothesize that the unique haplotype VIII originated in the lower South Island, and that its spread northwards was obstructed by the opening of Cook Strait approximately 15 000-16 000 years ago and the subsequent establishment of present-day surface water circulation patterns in Greater Cook Strait. We suggest that present-day strong tidal flows and turbulent mixing of water masses in Cook Strait, and intense up-welling on the east and west coasts in this region, represent a barrier to gene flow between mussels located in the North Island and northern South Island vs. mussels in most of the South Island and Stewart Island.     

Translocation of 32P between interacting mycelia of a wood-decomposing fungus and ectomycorrhizal fungi in microcosm systems

Interactions between saprotrophic and ectomycorrhizal fungi have been largely ignored, although their mycelia often share the same microsites. The mycelial systems show general similarity to each other and, although the enzymatic potential of the saprotrophic fungi is generally considered to be higher, the importance of organic nutrient sources to ectomycorrhizal fungi is now widely accepted. In the experiments described here, nutritional interactions involving transfer of elements from one mycelium to the other have been monitored dynamically using radioactive tracers and a non-destructive electronic autoradiography system. Microcosms were used in which mycelial systems of the ectomycorrhizal fungi Suillus variegatus and Paxillus involutus , extending from Pinus sylvestris host plants, were confronted with mycelia of the saprotroph Hypholoma fasciculare extending from wood blocks. The fungi showed a clear morphological confrontation response. The mycorrhizal mycelium often formed dense patches over the Hypholoma mycelia. Up to 25% of the ³²P present in the Hypholoma mycelium was captured by the mycorrhizal fungi and translocated to the plant host within 30 d. The transfer of ³²P to the saprotroph from labelled mycorrhizal mycelium was one to two orders of magnitude lower. The significance of this transfer as a 'short cut' in nutrient cycling is discussed.     

Ceratophyllum demersum hampers phytoplankton development in some small Norwegian lakes over a wide range of phosphorus concentrations and geographical latitude

1. Data on submerged and floating-leafed macrophytes, phytoplankton, nutrients (N, P) and calcium were collected from twenty-four small lakes ( 1 km²) over a wide range of latitudes in Norway. The majority of the investigated lakes were mesotrophic or eutrophic, and most of the lakes were markedly affected by diffuse and point-source runoff from agriculture. According to their macrophyte species composition, the majority of the lakes can be classified as Potamogeton lakes or Chara lakes, or a combination of these.
2. This study is consistent with the 'two alternative stable states' hypothesis. We observed clearwater lakes with dense macrophyte cover over a wider range of total P concentration than has been reported previously: from 30 to more than 700 mg P m^–3. The clearwater state was only observed in lakes with mean depths of less than 1.9 m.
3. Most clear lakes with high cover of submerged vegetation showed indications of N limitation.
4. In this study nearly all the macrophyte-dominated lakes with P concentrations above 30 mg m^–3 had dense stands of Ceratophyllum demersum (L.). This indicates that Ceratophyllum may also play an important role in stabilizing and maintaining a clearwater state at high P concentrations.     

Demographic and genetic effects on reproduction as related to population size in a rare, perennial herb, Senecio integrifolius (Asteraceae)

Seed-set of the rare and threatened plant Senecio integrifolius increased significantly with population size. Experimental studies as well as field observations showed this to be due to density-dependent seed-set (Allee effect). Hand-pollination revealed lower seed-set, and a lower germination rate of inbred progeny than of outbred progeny, with great differences among populations. Contrary to general predictions in models of minimum viable population sizes, the present study indicates little negative effects of inbreeding in small populations. A genetic load model was invoked to explain the results, hypothesizing that purging of deleterious alleles in small populations has reduced inbreeding depression. However, no clear correlation between population size and genetic load was found. The results in this paper suggest that demographic and environmental factors are of greater immediate importance than population genetics in determining extinction probabilities of small plant populations.     

Seasonal Effects on Photosynthetic Electron Transport and Fluorescence Properties in Isolated Chloroplasts of Pinus silvestris

Chloroplasts were isolated from primary needles of 1-year-old seedlings and from secondary needles of a 20-year-old pine tree in a natural stand. In autumn the electron transport capacities of PSII, PSI and PS (II + I) decreased and the electron transport between PSII and PSI became inhibited in October in the 20-year-old tree. This inhibition lasted until May the following year. The partial reactions of PSI and PSII still showed low but fairly constant rates during the whole winter seedlings. Seasonal changes in the electron transport properties of 1-year-old showed the same general trends as observed in the 20-year-old tree, but the changes were less pronounced. However, in snow-covered seedlings the PSI-mediated electron transport and the electron transport from H₂O to NADP increased during the late winter when the seedlings were still covered by snow. The total chlorophyll content of the needles decreased in autumn and winter. Low temperature fluorescence ratios of F692/F680 and F726/F680 indicated more severe destruction of the chlorophyll a antennae closely associated with the two photosystems than of the light harvesting chlorophyll a/b complex. In this case, too, the changes were more pronounced in the 20-year-old tree than in the 1-year-old seedlings. The chlorophyll/P700 ratios indicated a more marked reduction in the reaction centre molecules during autumn than in the antennae chlorophyll molecules. The changes in electron transport and low temperature fluorescence properties which occurred during autumn and winter were mainly reversed during spring.     

Lipid A, the active part of bacterial endotoxins in inducing serum colony stimulating activity and proliferation of splenic granulocyte/macrophage progenitor cells.

An analysis of which component of lipopolysaccharides (LPS), the lipid or the polysaccharide (PS), is active in stimulating the murine granulopoietic system has been performed. LPS with different structures, isolated from different mutant strains of Salmonella and chemical degradation products of lipopolysaccharides have been used. Lipid A obtained by acid hydrolysys of the LPS and complexed to bovine serum albumin (BSA) (lipid A-BSA) was shown to be active in generating serum colony stimulating factor (CSF) and in increasing the splenic colony forming cells (CFC) levels, although it was less active than the parent LPS. The polysaccharide (PS) showed no significant activity at the concentrations used. LPS (glycolipids) from R mutants of Salmonella minnesota were active to the same extent as the LPS. The fact that even the most defective LPS from the R mutant R595 which contains lipid A and KDO only is a potent endotoxin, points unequivocally, to lipid A, as the active principle in stimulating the granulopoietic system.