Overexpression of extracellular superoxide dismutase has a protective role against hyperoxia-induced brain injury in neonatal mice

There is increasing evidence that hyperoxia, particularly at the time of birth, may result in neurological injury, in particular to the susceptible vasculature of these tissues. This study was aimed at determining whether overexpression of extracellular superoxide dismutase (EC-SOD) is protective against brain injury induced by hyperoxia. Transgenic (TG) mice (with an extra copy of the human extracellular superoxide dismutase gene) and wild-type (WT) neonate mice were exposed to hyperoxia (95% of F(i) o(2) ) for 7 days after birth versus the control group in room air. Brain positron emission tomography (PET) scanning with fludeoxyglucose (FDG) isotope uptake was performed after exposure. To assess apoptosis induced by hyperoxia exposure, caspase 3 ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were performed. Quantitative western blot for the following inflammatory markers was performed: glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, macrophage-inhibiting factor, and phospho-AMP-activated protein kinase. PET scanning with FDG isotope uptake showed significantly higher uptake in the WT hyperoxia neonate brain group (0.14 ± 0.03) than in both the TG group (0.09 ± 0.01) and the control group (0.08 ± 0.02) (P< 0.05). Histopathological investigation showed more apoptosis and dead neurons in hippocampus and cerebellum brain sections of WT neonate mice after exposure to hyperoxia than in TG mice; this finding was also confirmed by TUNEL staining. The caspase 3 assay confirmed the finding of more apoptosis in WT hyperoxia neonates (0.814 ± 0.112) than in the TG hyperoxic group (0.579 ± 0.144) (P < 0.05); this finding was also confirmed by TUNEL staining. Quantitative western blotting for the inflammatory and metabolic markers showed significantly higher expression in the WT group than in the TG and control groups. Thus, overexpression of EC-SOD in the neonate brain offers significant protection against hyperoxia-induced brain damage.     

Monoclonal antibodies reveal changes in predator efficiency with prey spatial pattern

Spatially explicit predator–prey interactions can alter the predatory potential of natural enemies augmented through conservation biological control. To test hypotheses regarding such interactions and predatory efficiency, we used a combination of molecular techniques and mark–release–recapture to study the foraging behaviour of a generalist carabid predator, Poecilus cupreus , in response to spatial patterns of its cereal aphid prey ( Metapolophium dirhodum and Sitobion avenae ). Beetle and aphid numbers were measured across two grids of sampling locations, within which aphid spatial pattern had been manipulated to generate patchy and more homogenous distributions. Aphid consumption was measured by enzyme-linked immunosorbent assays (ELISA) of beetle gut contents, using an aphid-specific monoclonal antibody. Movement and distribution patterns suggest that P. cupreus does not aggregate at, nor instigate prey-taxis within, aphid patches. However, more than two-thirds of the 2169 P. cupreus tested by ELISA had consumed aphids and the proportion of beetles containing aphid proteins was positively related to aphid density. Against expectation, the proportion of predators feeding on aphids was greatest where prey were homogenously distributed, and this was attributed to the loss of partial refuges for prey in aphid patches. The functional value of this type of uniform foraging strategy is ideally suited to early colonization of the crop habitat, when aphid numbers are low, before populations build up and form strong spatial patterns.     

Changes in fish assemblages following 10 years of protection in Tasmanian marine protected areas

Most studies examining effects of marine protected areas (MPAs) on fish assemblages are potentially confounded, either because they are once off comparisons between fished and unfished locations, or because they are snapshot studies over a fixed period. Here we compare long-term changes within fully protected Tasmanian marine reserves with changes at external reference sites on an annual basis over the first ten years of protection. The results highlight the importance of long-term datasets for differentiating changes occurring over differing time scales. Notable results include a statistically significant increase in abundance of Latridopsis forsteri and large fish (> 300 mm) when examined across all reserves relative to controls, and a 10-fold increase in the abundance of large fish and a doubling of per site species richness of large fish within the Tinderbox Marine Reserve relative to controls. Short-term resident species that recruit sporadically show very different patterns in reserves compared to those that recruit regularly and have long-term age-class storage. While several recent reviews have suggested size of MPAs and duration of protection has little influence on the extent of recovery, our results suggest this is not the case and that responses can be slow, complex and species-specific. The extent of localised fishing pressure appeared to have a substantial influence on the degree of change detected, potentially confounding meta-analyses of recovery rates in MPAs if overlooked as a relevant parameter.     

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K _m ^I– =2.5 mM; k _cat ^I– =462 s^–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K _m ^I– =18.1 mM; k _cat ^I– =38 s^–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804.     

Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH₂-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K_m of 17.2 (± 0.48) μM for DMS (k_cat = 5.45 s⁻¹) and a V_max of 1.25 (± 0.01) μmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²⁺, Cd²⁺, and Pb²⁺ ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH₂-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6, 7, 34), with specific activities around 30 nmol NADH oxidized min⁻¹ mg protein⁻¹. The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO₂). During growth on DMSO₂, H. sulfonivorans first reduces DMSO₂ to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS monooxygenase. Oxidation of methanethiol to formaldehyde by methanethiol oxidase yields another mole of formaldehyde, which is either assimilated into biomass or oxidized to carbon dioxide to provide reducing equivalents (Fig. ​(Fig.1).1). DMS monooxygenase activity is present in the soluble protein fraction during growth on these compounds (6, 7). A 53-kDa polypeptide was previously observed in organisms grown on DMS, DMSO, and DMSO₂ (6, 7), but its significance in the metabolism of these compounds was unknown.Open in a separate windowFIG. 1.Pathway and enzymes of dimethylsulfone degradation in Hyphomicrobium sulfonivorans S1. Reduction of dimethylsulfone [DMSO₂; (CH₃)₂SO₂] to dimethyl sulfoxide [DMSO; (CH₃)₂SO] and further reduction of DMSO to dimethylsulfide provides the substrate for DMS monooxygenase. Formaldehyde is either assimilated (via the serine cycle) or oxidized to CO₂ providing reducing equivalents. Sulfide is oxidized to sulfate; see reference 7 for further details.     

A phospho-proteomic screen identifies novel S6K1 and mTORC1 substrates revealing additional complexity in the signaling network regulating cell growth

S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70^S6K1 isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85^S6K1 isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85^S6K1 substrates. Four novel putative p85^S6K1 substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.     

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.     

Functional Diversity of Plant–Pollinator Interaction Webs Enhances the Persistence of Plant Communities

Pollination is exclusively or mainly animal mediated for 70% to 90% of angiosperm species. Thus, pollinators provide an essential ecosystem service to humankind. However, the impact of human-induced biodiversity loss on the functioning of plant–pollinator interactions has not been tested experimentally. To understand how plant communities respond to diversity changes in their pollinating fauna, we manipulated the functional diversity of both plants and pollinators under natural conditions. Increasing the functional diversity of both plants and pollinators led to the recruitment of more diverse plant communities. After two years the plant communities pollinated by the most functionally diverse pollinator assemblage contained about 50% more plant species than did plant communities pollinated by less-diverse pollinator assemblages. Moreover, the positive effect of functional diversity was explained by a complementarity between functional groups of pollinators and plants. Thus, the functional diversity of pollination networks may be critical to ecosystem sustainability.     

Ribosomal Protein Genes RPS10 and RPS26 Are Commonly Mutated in Diamond-Blackfan Anemia

Diamond-Blackfan anemia (DBA), an inherited bone marrow failure syndrome characterized by anemia that usually presents before the first birthday or in early childhood, is associated with birth defects and an increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart, and urinary system defects that are present in ∼30%–50% of patients. DBA has been associated with mutations in seven ribosomal protein (RP) genes, RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, and RPS7, in about 43% of patients. To continue our large-scale screen of RP genes in a DBA population, we sequenced 35 ribosomal protein genes, RPL15, RPL24, RPL29, RPL32, RPL34, RPL9, RPL37, RPS14, RPS23, RPL10A, RPS10, RPS12, RPS18, RPL30, RPS20, RPL12, RPL7A, RPS6, RPL27A, RPLP2, RPS25, RPS3, RPL41, RPL6, RPLP0, RPS26, RPL21, RPL36AL, RPS29, RPL4, RPLP1, RPL13, RPS15A, RPS2, and RPL38, in our DBA patient cohort of 117 probands. We identified three distinct mutations of RPS10 in five probands and nine distinct mutations of RPS26 in 12 probands. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knockdown of RPS10 or RPS26 expression with siRNAs, which indicates that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins in rRNA processing.     

Fibroblasts derived from long‐lived insulin receptor substrate 1 null mice are not resistant to multiple forms of stress

Reduced signalling through the insulin/insulin-like growth factor-1 signalling (IIS) pathway is a highly conserved lifespan determinant in model organisms. The precise mechanism underlying the effects of the IIS on lifespan and health is currently unclear, although cellular stress resistance may be important. We have previously demonstrated that mice globally lacking insulin receptor substrate 1 (Irs1^−/−) are long-lived and enjoy a greater period of their life free from age-related pathology compared with wild-type (WT) controls. In this study, we show that primary dermal fibroblasts and primary myoblasts derived from Irs1^−/− mice are no more resistant to a range of oxidant and nonoxidant chemical stressors than cells derived from WT mice.