Spatial variation of the stable nitrogen isotope ratio of woody plants along a topoedaphic gradient in a subtropical savanna

Variation in the stable N isotope ratio (δ¹⁵N) of plants and soils often reflects the influence of environment on the N cycle. We measured leaf δ¹⁵N and N concentration ([N]) on all individuals of Prosopis glandulosa (deciduous tree legume), Condalia hookeri (evergreen shrub), and Zanthoxylum fagara (evergreen shrub) present within a belt transect 308 m long × 12 m wide in a subtropical savanna ecosystem in southern Texas, USA in April and August 2005. Soil texture, gravimetric water content (GWC), total N and δ¹⁵N were also measured along the transect. At the landscape scale, leaf δ¹⁵N was negatively related to elevation for all the three species along this topoedaphic sequence. Changes in soil δ¹⁵N, total N, and GWC appeared to contribute to this spatial pattern of leaf δ¹⁵N. In lower portions of the landscape, greater soil N availability and GWC are associated with relatively high rates of both N mineralization and nitrification. Both soil δ¹⁵N and leaf [N] were positively correlated with leaf δ¹⁵N of non-N₂ fixing plants. Leaf δ¹⁵N of P. glandulosa, an N₂-fixing legume, did not correlate with leaf [N]; the δ¹⁵N of P. glandulosa’s leaves were closer to atmospheric N₂ and significantly lower than those of C. hookeri and Z. fagara. Additionally, at smaller spatial scales, a proximity index (which reflected the density and distance of surrounding P. glandulosa trees) was negatively correlated with leaf δ¹⁵N of C. hookeri and Z. fagara, indicating the N₂-fixing P. glandulosa may be important to the N nutrition of nearby non-N₂-fixing species. Our results indicate plant ¹⁵N natural abundance can reflect the extent of N retention and help us better understand N dynamics and plant-soil interactions at ecosystem and landscape scales.     

TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

Introduction

The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling.

Methods

Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism.

Results

While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro.

Discussion

TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.     

Progressive defects observed in mouse sperm during the course of three generations of selenium deficiency

Three successive generations of mice were fed a Torula yeast based Se-deficient diet with or without 0.1 ppm Se in the drinking water. The Se-deficient mice, in the course of three generations, showed a decrease in body weight, testis weight, epididymal weight, and sperm production. The percentage of morphologically abnormal sperm increased in successive generations. The majority of sperm defects were found in the midpiece region of the tail. Many of these aberrant sperm were motile. A progressive decrease in fertility was noted during the first two generations of Se deficiency. This system confirms the essential role of Se in spermatogenesis and provides a model for the evaluation of the primary effect of Se deprivation on the structural development of sperm.     

Synergistic activation of ENaC by three membrane-bound channel-activating serine proteases (mCAP1, mCAP2, and mCAP3) and serum- and glucocorticoid-regulated kinase (Sgk1) in Xenopus Oocytes

Sodium balance is maintained by the precise regulation of the activity of the epithelial sodium channel (ENaC) in the kidney. We have recently reported an extracellular activation of ENaC-mediated sodium transport (I(Na)) by a GPI-anchored serine protease (mouse channel-activating protein, mCAP1) that was isolated from a cortical collecting duct cell line derived from mouse kidney. In the present study, we have identified two additional membrane-bound serine proteases (mCAP2 and mCAP3) that are expressed in the same cell line. We show that each of these proteases is able to increase I(Na) 6-10-fold in the Xenopus oocyte expression system. I(Na) and the number (N) of channels expressed at the cell surface (measured by binding of a FLAG monoclonal I(125)-radioiodinated antibody) were measured in the same oocyte. Using this assay, we show that mCAP1 increases I(Na) 10-fold (P < 0.001) but N remained unchanged (P = 0.9), indicating that mCAP1 regulates ENaC activity by increasing its average open probability of the whole cell (wcP(o)). The serum- and glucocorticoid-regulated kinase (Sgk1) involved in the aldosterone-dependent signaling cascade enhances I(Na) by 2.5-fold (P < 0.001) and N by 1.6-fold (P < 0.001), indicating a dual effect on N and wcP(o). Compared with Sgk1 alone, coexpression of Sgk1 with mCAP1 leads to a ninefold increase in I(Na) (P < 0.001) and 1.3-fold in N (P < 0.02). Similar results were observed for mCAP2 and mCAP3. The synergism between CAPs and Sgk1 on I(Na) was always more than additive, indicating a true potentiation. The synergistic effect of the two activation pathways allows a large dynamic range for ENaC-mediated sodium regulation crucial for a tight control of sodium homeostasis.     

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.     

STRUCTURALLY PRESERVED FOSSIL PLANTS FROM ANTARCTICA. I. ANTARCTICYCAS,GEN. NOV., A TRIASSIC CYCAD STEM FROM THE BEARDMORE GLACIER AREA

Silicified stems with typical cycadalean anatomy are described from specimens collected from the Fremouw Formation (Triassic) in the Transantarctic Mountains of Antarctica. Axes are slender with a large parenchymatous pith and cortex separated by a narrow ring of vascular tissue. Mucilage canals are present in both pith and cortex. Vascular tissue consists of endarch primary xylem, a narrow band of secondary xylem tracheids, cambial zone, and region of secondary phloem. Vascular bundles contain uni- to triseriate rays with larger rays up to 2 mm wide separating the individual bundles. Pitting on primary xylem elements ranges from helical to scalariform; secondary xylem tracheids exhibit alternate circular bordered pits. Traces, often accompanied by a mucilage canal, extend out through the large rays into the cortex where some assume a girdling configuration. A zone of periderm is present at the periphery of the stem. Large and small roots are attached to the stem and are conspicuous in the surrounding matrix. The anatomy of the Antarctic cycad is compared with that of other fossil and extant cycadalean stems.     

Phosphorus and carbon availability regulate structural composition and complexity of AM fungal mycelium

The regulation of the structural composition and complexity of the mycelium of arbuscular mycorrhizal (AM) fungi is not well understood due to their obligate biotrophic nature. The aim of this study was to investigate the structure of extraradical mycelium at high and low availability of carbon (C) to the roots and phosphorus (P) to the fungus. We used monoxenic cultures of the AM fungus Rhizophagus irregularis (formerly Glomus intraradices) with transformed carrot roots as the host in a cultivation system including a root-free compartment into which the extraradical mycelium could grow. We found that high C availability increased hyphal length and spore production and anastomosis formation within individual mycelia. High P availability increased the formation of branched absorbing structures and reduced spore production and the overall length of runner hyphae. The complexity of the mycelium, as indicated by its fractal dimensions, increased with both high C and P availability. The results indicate that low P availability induces a growth pattern that reflects foraging for both P and C. Low C availability to AM roots could still support the explorative development of the mycelium when P availability was low. These findings help us to better understand the development of AM fungi in ecosystems with high P input and/or when plants are subjected to shading, grazing or any management practice that reduces the photosynthetic ability of the plant.     

Presence of chromosomal mosaicism in abnormal preimplantation embryos detected by fluorescence in situ hybridisation

The extent of chromosomal mosaicism in human preimplantation embryos was examined using an improved procedure for the preparation and spreading of interphase nuclei for use in fluorescence in situ hybridisation, allowing the analysis of every nucleus within an embryo. One cell showed no hybridisation signals in only three of the 38 embryos that were included in this study, i.e. the hybridisation efficiency per successfully spread nucleus was 99% (197/200). Double-target in situ hybridisation analyses with X- and Y-chromosome-specific probes was performed to analyse nine embryos resulting from normal fertilisation, 22 polypronucleate embryos and seven cleavage-stage embryos where no (apronucleate) or only one pronucleus (monopronucleate) was observed. We also analysed autosomes 1 and 7 by double-target in situ hybridisation in the nuclei of two apronucleate, one monopronucleate and four polypronucleate embryos. All nine embryos that resulted from normal fertilisation were uniformly XY or XX. None of the apronucleate or monopronucleate embryos was haploid: three were diploid, one was triploid and three were mosaic. Fertilisation was detected by the presence of a Y-specific signal in four of these embryos. Of the polypronucleate embryos, two were diploid, two were triploid and 18 were mosaic for the sex chromosomes and/or autosomes 1 and 7. These results demonstrate that fertilisation sometimes occurs in monopronucleate embryos and that chromosomal mosaicism can be detected with high efficiency in apronucleate, monopronucleate and polypronucleate human embryos using fluorescence in situ hybridisation.     

Stereochemistry of the hydrolysis of α,α-trehalose by trehalase,determined by using a labelled substrate

(1,1′-¹³C)α,α-Trehalose was obtained in 37% yield from the Pavia condensation of 2,3,4,6-tetra-O-benzyl-d-(1-¹³C)glucopyranose, in dichloromethane in the presence of trifluoromethanesulfonic anhydride, followed by the usual deprotection techniques. The hydrolysis of this substrate by cockchafer trehalase was monitored at 37° by using ¹³C-n.m.r. spectroscopy with short recording times. Equimolecular amounts of α- and β-d-glucopyranose are released simultaneously by the action of the enzyme. This result is consistent with a bimolecular substitution mechanism, taking into account previous results involving C-2 asymmetric participation in the catalytic step of hydrolysis of α,α-trehalose. For comparative evaluation of its accuracy, the usual polarimetric technique was also used for the determination of the anomeric configuration of the d-glucose released by the action of the enzyme on α,α-trehalose.     

Normal levels of zinc and sulfhydryls in morphologically abnormal populations of spermatozoa from moderately zinc-deficient rats

Zinc is required for spermatogenesis in mammals and is concentrated in the dense outer fibers of the sperm tail, where it is associated with cysteine-rich protein. To investigate the effects of marginal zinc deficiency upon dense fiber formation and upon sperm quality in general, weanling Sprague-Dawley rats were administered a commercial low-zinc diet, supplemented with phytate, for approximately 60 days, and were compared with controls fed the same diet plus 50 ppm zinc in their drinking water. The following characteristics of the zinc-deficient rats were significantly lower than in the controls: body weight, testis weight, epididymis weight, seminal vesicle weight, sperm content of the cauda epididy-midis, sperm motility, testis zinc, and hair zinc. By contrast, the levels of sperm zinc and sperm sulfhydryls were the same in the zinc-deficient and control rats. The zinc-deficient rats displayed a highly variable spectrum of sperm defects, which included decapitation, disorganized and redundant tail elements, and superfluous cytoplasm. However, abortive dense fiber development was only rarely observed. Apparently, even when availability of zinc is limited and reduced sperm production ensues, elaboration of dense fibers rich in zinc and sulfhydryls continues to be obligatory for the completion of spermiogenesis.