Patterns of natural 15N abundance in the leaf-to-soil continuum of tropical rain forests differing in N availability on Mount Kinabalu,Borneo

We investigated the natural abundance of ¹⁵N in sun leaves and other components of tropical rain forests on altitudinal sequences of eight sites that form a gradient of soil N availability with varying ectomycorrhizal abundances on Mt. Kinabalu, Borneo. We investigated how soil N availability and ectomycorrhizal abundance related to the ¹⁵N abundance of ecosystem components. ¹⁵N values (¹⁵N abundance relative to ¹⁴N) increased consistently in the following order at each site: sun leaves, leaf litter, fine roots and from shallower organic to deeper mineral soil horizons. Enrichment (3–6 ¹⁵N) of ¹⁵N occurred at the litter–topsoil interface at all sites, and the magnitude of the enrichment correlated negatively with ¹⁵N depletion in the foliage, irrespective of ectomycorrhizal abundance. Foliar ¹⁵N values significantly positively correlated with their N concentrations. Foliar (and litter and root) ¹⁵N values correlated positively with NO₃ availability, and negatively with NH₄ availability. The two positive correlations of foliar ¹⁵N with foliar N and NO₃ availability were inconsistent with the assumption that stronger nitrification (hence a greater nitrate availability) produced a more ¹⁵N-depleted active inorganic N pool. The isotopic fractionation during the passage of N through ectomycorrhizas to plants might explain the positive correlation of foliar ¹⁵N and N concentration; however, this mechanism could not fully explain the correlation in our case because strong foliar ¹⁵N depletions occurred at the sites that lacked ectomycorrhizas. Alternatively, the positive correlation across sites reflected the tightness of N cycling. Strong nitrification and associated isotopic fractionation might have occurred at N-richer sites and the subsequent removal of NO₃ from the system could decrease isotopically `lighter' N at these sites.     

Characterization of the 5-Carboxyvanillate Decarboxylase Gene and Its Role in Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6

Sphingomonas paucimobilis SYK-6 degrades a lignin-related biphenyl compound, 5,5′-dehydrodivanillate (DDVA), to 5-carboxyvanillate (5CVA) by the enzyme reactions catalyzed by the DDVA O-demethylase (LigX), the ring cleavage oxygenase (LigZ), and the meta-cleavage compound hydrolase (LigY). In this study we examined the degradation step of 5CVA. 5CVA was transformed to vanillate, O-demethylated, and further degraded via the protocatechuate 4,5-cleavage pathway by this strain. A cosmid clone which conferred the 5CVA degradation activity to a host strain was isolated. In the 7.0-kb EcoRI fragment of the cosmid we found a 1,002-bp open reading frame responsible for the conversion of 5CVA to vanillate, and we designated it ligW. The gene product of ligW (LigW) catalyzed the decarboxylation of 5CVA to produce vanillate along with the specific incorporation of deuterium from deuterium oxide, indicating that LigW is a nonoxidative decarboxylase of 5CVA. LigW did not require any metal ions or cofactors for its activity. The decarboxylase activity was specific to 5CVA. Inhibition experiments with 5CVA analogs suggested that two carboxyl groups oriented meta to each other in 5CVA are important to the substrate recognition by LigW. Gene walking analysis indicated that the ligW gene was located on the 18-kb DNA region with other DDVA catabolic genes, including ligZ, ligY, and ligX.     

Expression of inducible nitric oxide (NO) synthase but not prevention by its gene ablation of hepatocarcinogenesis with fibrosis caused by a choline-deficient, L-amino acid-defined diet in rats and mice.

Expression of inducible nitric oxide synthase (iNOS) and effects of iNOS gene ablation on the hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, were examined in male F344 rats and C57BL/6J wild-type and iNOS-/- mice. Western blot, RT-PCR and immunohistochemical analyses revealed increased expression of iNOS protein and mRNA in the livers of rats and wild-type mice fed a CDAA diet for 12-80 weeks, associated with elevated serum NO(x) and liver nitrotyrosine levels. iNOS-/- mice demonstrated greater liver injury and fibrosis in the early stage than their wild-type counterparts, but this did not significantly affect the incidence and multiplicity of altered foci, adenomas and hepatocellular carcinomas in spite of immunohistochemical iNOS expression in these lesions. Results suggested no major determinant roles of the expressed iNOS in the development of liver tumors caused by the CDAA diet.     

PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans

Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress.     

Cloning of structural gene of Deinococcus radiodurans UV-endonuclease beta

To characterize its enzymic property we cloned and sequenced the gene of Deinococcus radiodurans encoding UV-endonuclease beta, an alternative enzyme to UvrABC repairing damaged DNA. Amino acid substitutions were found in UV-sensitive mutants. The putative amino acid sequence had some similarity with those of eukaryotic UV-endonucleases and with a sequence found in a protein data base of Bacillus subtilis.     

KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.     

Metabolite annotations based on the integration of mass spectral information

A large number of metabolites are found in each plant, most of which have not yet been identified. Development of a methodology is required to deal systematically with unknown metabolites, and to elucidate their biological roles in an integrated 'omics' framework. Here we report the development of a 'metabolite annotation' procedure. The metabolite annotation is a process by which structures and functions are inferred for metabolites. Tomato ( Solanum lycopersicum cv. Micro-Tom) was used as a model for this study using LC-FTICR-MS. Collected mass spectral features, together with predicted molecular formulae and putative structures, were provided as metabolite annotations for 869 metabolites. Comparison with public databases suggests that 494 metabolites are novel. A grading system was introduced to describe the evidence supporting the annotations. Based on the comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that are frequently found in tomato fruit tissues, and predicted novel metabolic pathways for flavonoids and glycoalkaloids. These results demonstrate that metabolite annotation facilitates the systematic analysis of unknown metabolites and biological interpretation of their relationships, which provide a basis for integrating metabolite information into the system-level study of plant biology.     

Requirement for the p75 TNF-alpha receptor 2 in the regulation of airway hyperresponsiveness by gamma delta T cells

In a recent study, we found that TNF-alpha negatively regulates airway responsiveness through the activation of gammadelta T cells. The biological activities of TNF-alpha are mediated by two structurally related but functionally distinct receptors, p55 (TNFR1) and p75 (TNFR2), which are independently expressed on the cell surface. However, the relative importance of either TNFR in airway hyperresponsiveness (AHR) is unknown. To investigate the importance of these TNFRs in the development of allergen-induced AHR, p55-deficient and p75-deficient mice were sensitized to OVA by i.p. injection and subsequently challenged with OVA via the airways; airway responsiveness to inhaled methacholine was monitored. p75-deficient mice developed AHR to a similar degree as control mice. In contrast, p55-deficient mice, which were sensitized and challenged with OVA, failed to develop AHR. In p55-deficient mice, both the numbers of eosinophils and levels of IL-5 in bronchoalveolar lavage fluid were significantly lower than in sensitized/challenged control mice (p < 0.05). However, depletion of gammadelta T cells resulted in significant increases in AHR in the p55-deficient mice, whereas no significant effect of gammadelta T cell depletion was evident in the p75-deficient mice. These data indicate that, in the absence of TNFR1 (p55), where TNF-alpha uses the p75 pathway exclusively, the development of AHR is regulated by gammadelta T cells.     

Isozymes of Superoxide Dismutase in Chlamydomonas and Purification of One of the Major Isozymes Containing Fe

Electrophoretic analysis of Chlamydomonas reinhardtii extractrevealed at least 4 distinct superoxide dismutase (SOD) activitybands as well as several additional minor bands. Among them,one was deduced to be Fe-type and the other three Mn-type basedon their susceptibility to KCN and H₂O₂. The Fe-SOD, which occupiedabout 40% of the total soluble activity, was purified to homogeneityusing ammonium sulfate fractionation followed by DEAE-cellulose,hydroxyapatite, and Superdex 75 gel-permeation chromatography.The 40-kDa native enzyme was composed of two identical 20-kDasubunits with a low shoulder of absorption at {small tilde}350nm. The NH₂-terminal amino acid sequence determined up to residue29 showed a high homology to those of Fe-SOD from Arabidopsisthaliana, Glycine max, and Nicotiana plumbaginifolia. (Received December 21, 1992; Accepted May 28, 1993)