The cell adhesion molecule neuroplastin-65 inhibits hippocampal long-term potentiation via a mitogen-activated protein kinase p38-dependent reduction in surface expression of GluR1-containing glutamate receptors

Neuroplastin-65 is a brain-specific, synapse-enriched member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. Previous studies highlighted the importance of neuroplastin-65 for long-term potentiation (LTP), but the mechanism was unclear. Here, we show how neuroplastin-65 activation of mitogen-activated protein kinase p38 (p38MAPK) modified synapse strength by altering surface glutamate receptor expression. Organotypic hippocampal slice cultures treated with the complete extracellular fragment of neuroplastin-65 (FcIg1-3) sustained an increase in the phosphorylation of p38MAPK and an inability to induce LTP at hippocampal synapses. The LTP block was reversed by application of the p38MAPK inhibitor SB202190, suggesting that p38MAPK activation occurred downstream of neuroplastin-65 binding and upstream of the loss of LTP. Further investigation revealed that the mechanism underlying neuroplastin-65-dependent prevention of LTP was a p38MAPK-dependent acceleration of the loss of surface-exposed glutamate receptor subunits that was reversed by pretreatment with the p38MAPK inhibitor SB202190. Our results indicate that neuroplastin-65 binding and associated stimulation of p38MAPK activity are upstream of a mechanism to control surface glutamate receptor expression and thereby influence plasticity at excitatory hippocampal synapses.     

Cereal density and N-fertiliser effects on the flora and biodiversity value of arable headlands

Modern intensive farming caused pronounced changes to the European arable flora. Many species adapted to less intensive traditional farming declined severely, as did the potential of unsown arable vegetation to support higher trophic levels. To reverse these trends, various agri-environment measures were introduced. One such measure is to manage cereal headlands as conservation headlands, involving strict restrictions on pesticide and fertiliser use. An additional modification to management which could reduce crop competition and thus deliver benefits to arable plants is cereal sowing at reduced rates. However, little is known about its benefits to rare and declining arable plants, or to species of value to higher trophic levels, and whether it can be implemented without concomitant increase in undesirable weeds. We set up identical two-factorial experiments in winter wheat and spring barley, combining a nitrogen fertiliser versus no fertiliser treatment with cereal sowing at economic rates versus sowing at rates reduced by 75 %, with added sowing of a mixture of rare arable species. Both experiments also included an uncropped but cultivated control equivalent to another agri-environment measure. Our results show that reduced cereal sowing in conservation headlands can benefit rare and declining species, as well as arable plant diversity, without necessarily resulting in a concomitant increase in undesirable weeds. While such benefits tended to be larger in uncropped cultivated controls, conservation headlands have the advantage of not requiring land being taken out of production. Moreover, as shown in this study, their benefits to arable plants can be maximised by reduced sowing.     

Aurora-A Kinase Is Essential for Bipolar Spindle Formation and Early Development

Aurora-A is a conserved kinase implicated in mitotic regulation and carcinogenesis. Aurora-A was previously implicated in mitotic entry and spindle assembly, although contradictory results prevented a clear understanding of the roles of Aurora-A in mammals. We developed a conditional null mutation in the mouse Aurora-A gene to investigate Aurora-A functions in primary cells ex vivo and in vivo. We show here that conditional Aurora-A ablation in cultured embryonic fibroblasts causes impaired mitotic entry and mitotic arrest with a profound defect in bipolar spindle formation. Germ line Aurora-A deficiency causes embryonic death at the blastocyst stage with pronounced cell proliferation failure, mitotic arrest, and monopolar spindle formation. Aurora-A deletion in mid-gestation embryos causes an increase in mitotic and apoptotic cells. These results indicate that murine Aurora-A facilitates, but is not absolutely required for, mitotic entry in murine embryonic fibroblasts and is essential for centrosome separation and bipolar spindle formation in vitro and in vivo. Aurora-A deletion increases apoptosis, suggesting that molecular therapies targeting Aurora-A may be effective in inducing tumor cell apoptosis. Aurora-A conditional mutant mice provide a valuable system for further defining Aurora-A functions and for predicting effects of Aurora-A therapeutic intervention.The equal partitioning of chromosomes at mitosis is critical for avoiding aneuploidy, a condition associated with spontaneous miscarriage, developmental disorders, and cancer (50). Mitosis requires coordinated completion of multiple events including nuclear envelope breakdown, chromosome condensation and congression to the metaphase plate, centrosome separation, spindle formation, chromosome-spindle attachment and error correction, sister chromatid separation, and cytokinesis. Multiple regulators, many of which are kinases, are required to ensure that each event is completed in a timely fashion and in the proper order (reviewed in reference 46). Although a number of mitotic kinases have been identified, their targets and the intricacies of mitotic signal transduction pathways are just beginning to be understood.The Aurora kinases are key mitotic regulators in eukaryotes (reviewed in reference 45). The Aurora family includes a single member in yeasts (Saccharomyces cerevisiae Ipl1p, Schizosaccharomyces pombe Ark1), two members each in Caenorhabditis elegans and Drosophila, and two or three members in vertebrates. Although originally given a variety of names, Aurora kinases in multicellular eukaryotes have subsequently been classified into A, B, and C groups based on patterns of mitotic subcellular localization and homology, which also appear to reflect functional distinctions (8, 46). Aurora-A kinases are observed at centrosomes and adjacent spindle fibers, and current evidence supports key roles in regulating protein localization and function at centrosomes, as well as regulation of the assembly, stability, and function of the mitotic spindle (reviewed in reference 43). Aurora-B kinases display “chromosomal passenger” localization, residing on mitotic chromosomes and subsequently moving to the spindle midzone after separation of sister chromatids. Aurora-B family members have been implicated in the regulation of kinetochore-spindle attachment, the spindle checkpoint, and cytokinesis (reviewed in references 1 and 8). Aurora-C kinases, which have only been identified in mammals, have a limited expression pattern and appear to have functions that overlap those of Aurora-B (7, 53).The human Aurora-A kinase (hAurA) was first identified because of its overexpression in cancer cell lines (5, 58). The hAurA gene (stk15) resides on chromosome 20q13, a region frequently amplified in human cancers (5, 58). hAurA has been dubbed an oncogene because of the fact that its overexpression transforms immortalized rodent fibroblasts (5, 70). Polymorphisms in hAurA are associated with an increased risk of colon cancer, while murine AurA (mAurA) polymorphisms confer increased susceptibility to experimentally induced skin tumors (14). The mAurA gene is frequently amplified in radiation-induced lymphomas from p53 heterozygous mice, while loss of one mAurA allele has been observed in lymphomas from p53-null mice (41). Thus, aberrant AurA expression is associated with tumorigenesis, suggesting that insight into AurA functions will lead to a better understanding of tumorigenesis mechanisms.A number of experimental observations suggest that AurA kinases are required for normal centrosome maturation and bipolar spindle assembly. The AurA ortholog in Drosophila melanogaster (Aurora) was identified in a screen for mutations that impact the centrosome cycle (21). Syncytial embryos from hypomorphic Aurora mutant females display a variety of mitotic abnormalities resulting from a failure to separate centrosomes. Aurora-null flies die at the larval stage with characteristic monopolar spindles and circular chromosome arrays in larval neuroblasts. Such monopolar spindles arise from failed centrosome separation (21). Subsequent studies of Drosophila Aurora mutant alleles revealed additional defects in centrosome maturation (including a failure to localize transforming acidic coiled-coil protein, centrosomin, and γ-tubulin at centrosomes) and in asymmetric localization of Numb protein in sensory organ precursor cells (3, 17). Similar to the case in Drosophila, disruption of the C. elegans AurA ortholog AIR-1 by RNA interference (RNAi) or mutation causes defects in centrosome maturation and monopolar spindle formation. Centrosomes undergo normal separation but collapse, leading to monopolar spindle formation (16, 24, 56). Studies of the Xenopus AurA homolog pEg2 revealed similar phenotypes after overexpression of kinase-dead mutants, antibody-mediated inhibition, or immunodepletion (18, 19, 38, 52). Furthermore, Xenopus AurA has been shown to interact with and phosphorylate Eg5, a mitotic kinesin required for bipolar spindle formation, suggesting a possible mechanism by which AurA could influence bipolar spindle formation and/or stabilization (19). Thus, existing reports from these systems are quite consistent in implicating AurA in centrosome separation and function.In contrast to the systems described above, published reports of RNAi-mediated reduction of AurA expression in mammalian cell lines have contained conflicting results about the role of AurA in mitotic entry, bipolar spindle formation, and mitotic progression. AurA RNAi in HeLa cells was reported to block or delay mitotic entry, prompting the conclusion that AurA is essential for mitotic commitment in mammalian cells (27, 36). In contrast, other AurA RNAi studies showed accumulation of mitotic cells with monopolar spindles (12, 20, 67). These discrepancies call into question the functional conservation of AurA in mammals and highlight a need for additional studies to definitively address the roles of AurA. This is particularly critical for understanding the roles of AurA in cancer and for projecting possible effects of AurA inhibitors currently in development as anticancer agents. We used gene targeting in mouse embryonic stem (ES) cells to produce a conditional null allele at the AurA locus. Here we describe cellular phenotypes of AurA deletion in primary cells in vitro and developmental phenotypes of AurA mutant mice. We show that AurA deletion in primary embryonic fibroblasts causes delayed mitotic entry with accumulation of cells in early prophase, consistent with a role for AurA in mitotic entry. Nevertheless, AurA-deficient cells that enter prometaphase arrest with monopolar spindles and eventually exit mitosis without segregating their chromosomes. Prolonged culture of AurA-deficient cells leads to polyploidy with abnormal nuclear structure. Germ line AurA deficiency causes embryonic death at the blastocyst stage with mitotic arrest and monopolar spindle formation, while AurA deletion in mid-gestation embryos causes an increased mitotic index and increased apoptosis. Together, our findings indicate that AurA is required for timely mitotic entry and bipolar spindle formation in vitro and in vivo.     

Bacterial and fungal communities in bulk soil and rhizospheres of aluminum-tolerant and aluminum-sensitive maize (Zea mays L.) lines cultivated in unlimed and limed Cerrado soil

Liming of acidic soils can prevent aluminum toxicity and improve crop production. Some maize lines show aluminum (Al) tolerance, and exudation of organic acids by roots has been considered to represent an important mechanism involved in the tolerance. However, there is no information about the impact of liming on the structures of bacterial and fungal communities in Cerrado soil, nor if there are differences between the microbial communities from the rhizospheres of Al-tolerant and Al-sensitive maize lines. This study evaluated the effects of liming on the structure of bacterial and fungal communities in bulk soil and rhizospheres of Al-sensitive and Al-tolerant maize (Zea mays L.) lines cultivated in Cerrado soil by PCR-DGGE, 30 and 90 days after sowing. Bacterial fingerprints revealed that the bacterial communities from rhizospheres were more affected by aluminum stress in soil than by the maize line (Al-sensitive or Al-tolerant). Differences in bacterial communities were also observed over time (30 and 90 days after sowing), and these occurred mainly in the Actinobacteria. Conversely, fungal communities from the rhizosphere were weakly affected either by liming or by the rhizosphere, as observed from the DGGE profiles. Furthermore, only a few differences were observed in the DGGE profiles of the fungal populations during plant development when compared with bacterial communities. Cloning and sequencing of 16S rRNA gene fragments obtained from dominant DGGE bands detected in the bacterial profiles of the Cerrado bulk soil revealed that Actinomycetales and Rhizobiales were among the dominant ribotypes.     

Diversity of nifH gene pools in the rhizosphere of two cultivars of sorghum (Sorghum bicolor) treated with contrasting levels of nitrogen fertilizer.

The diversity of nitrogen-fixing bacteria was assessed in the rhizospheres of two cultivars of sorghum (IS 5322-C and IPA 1011) sown in Cerrado soil amended with two levels of nitrogen fertilizer (12 and 120 kg ha(-1)). The nifH gene was amplified directly from DNA extracted from the rhizospheres, and the PCR products cloned and sequenced. Four clone libraries were generated from the nifH fragments and 245 sequences were obtained. Most of the clones (57%) were closely related to nifH genes of uncultured bacteria. NifH clones affiliated with Azohydromonas spp., Ideonella sp., Rhizobium etli and Bradyrhizobium sp. were found in all libraries. Sequences affiliated with Delftia tsuruhatensis were found in the rhizosphere of both cultivars sown with high levels of nitrogen, while clones affiliated with Methylocystis sp. were detected only in plants sown under low levels of nitrogen. Moreover, clones affiliated with Paenibacillus durus could be found in libraries from the cultivar IS 5322-C sown either in high or low amounts of fertilizer. This study showed that the amount of nitrogen used for fertilization is the overriding determinative factor that influenced the nitrogen-fixing community structures in sorghum rhizospheres cultivated in Cerrado soil.     

Striga hermonthica decreases photosynthesis in Zea mays through effects on leaf cell structure

Maize seedlings were grown in pots either with or without preconditionedseeds of the parasitic weed, Striga hermonthica. After between4 and 8 weeks, net photosynthesis in the leaves of maize plantsinfected with Striga decreased compared to leaves of uninfectedcontrol plants. The activities of four enzymes of photosyntheticmetabolism were, however, little affected by infection. A pulse-chaseexperiment using ¹⁴CO₂ showed that C₄ acids were the main earlyproducts of assimilation even when the rate of photosynthesiswas much decreased by infection, but more radio-activity appearedin glycine and serine than in leaves of healthy maize plants.Leaves of infected maize required longer to reach a steady rateof photosynthesis upon enclosure in a leaf chamber than leavesof uninfected plants after similar treatment. Electron microscopy of transverse sections of the leaves ofinfected maize indicated that the cell walls in the bundle sheathand vascular tissue were less robust than in leaves of healthyplants. The results suggest that infection with Striga causesan increase in the permeability of cell walls in the bundlesheath, leakage of CO₂ from the bundle sheath cells and decreasedeffectiveness of C₄ photosynthesis in host leaves. Key words: Zea mays, Striga hermonthica, photosynthesis, photorespiration, enzyme activity     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa

Enteric neurotransmitters can modulate the biodefensive functions of the intestinal mucosa, but their role in mucosal interactions with enteropathogens is not well defined. Here we tested the hypothesis that norepinephrine (NE) modulates interactions between enterohemorrhagic Escherichia coli O157:H7 (EHEC) and the colonic epithelium. Mucosal sheets from porcine distal colon were mounted in Ussing chambers. Drugs and an inoculum of either Shiga toxin-negative or -positive EHEC were added to the contraluminal and luminal bathing medium, respectively. After 90 min, adherent bacteria were quantified by an adherence assay and by immunohistochemical methods; short-circuit current (I(sc)) was measured continuously to assess changes in active ion transport. NE-treated tissues exhibited concentration-dependent increases in I(sc) and EHEC adherence. NE did not alter adherence of a rodent-adapted, noninfectious E. coli strain or two porcine-adapted non-O157 E. coli strains. The actions of NE on EHEC adherence but not I(sc) were prevented by the alpha-adrenergic antagonist yohimbine and the PKA activator Sp-8-bromoadenosine-3',5'-cyclic monophosphorothioate. Like NE, the PKA inhibitor Rp-8-bromoadenosine-3',5'-cyclic monophosphorothioate or indirectly acting sympathomimetic agents increased EHEC adherence. Nerve fibers immunoreactive for the NE-synthesizing enzymes tyrosine hydroxylase and dopamine beta-hydroxylase appeared to innervate the colonic epithelium. EHEC-like immunoreactivity on the colonic surface had the appearance of bacterial microcolonies and increased after NE treatment by a phentolamine-sensitive mechanism. Through interactions with alpha(2)-adrenergic receptors, NE appears to increase EHEC adherence to the colonic mucosa. Changes in sympathetic neural outflow may alter intestinal susceptibility to infection.