Multiple Scales of Temporal Variability in Ecosystem Metabolism Rates: Results from 2 Years of Continuous Monitoring in a Forested Headwater Stream

Headwater streams are key sites of nutrient and organic matter processing and retention, but little is known about temporal variability in gross primary production (GPP) and ecosystem respiration (ER) rates as a result of the short duration of most metabolism measurements in lotic ecosystems. We examined temporal variability and controls on ecosystem metabolism by measuring daily rates continuously for 2 years in Walker Branch, a first-order deciduous forest stream. Four important scales of temporal variability in ecosystem metabolism rates were identified: (1) seasonal, (2) day-to-day, (3) episodic (storm-related), and (4) inter-annual. Seasonal patterns were largely controlled by the leaf phenology and productivity of the deciduous riparian forest. Walker Branch was strongly net heterotrophic throughout the year with the exception of the open-canopy spring when GPP and ER rates were co-equal. Day-to-day variability in weather conditions influenced light reaching the streambed, resulting in high day-to-day variability in GPP particularly during spring (daily light levels explained 84% of the variance in daily GPP in April). Episodic storms depressed GPP for several days in spring, but increased GPP in autumn by removing leaves shading the streambed. Storms depressed ER initially, but then stimulated ER to 2–3 times pre-storm levels for several days. Walker Branch was strongly net heterotrophic in both years of the study, with annual GPP being similar (488 and 519 g O₂ m⁻² y⁻¹ or 183 and 195 g C m⁻² y⁻¹) but annual ER being higher in 2004 than 2005 (−1,645 vs. −1,292 g O₂ m⁻² y⁻¹ or −617 and −485 g C m⁻² y⁻¹). Inter-annual variability in ecosystem metabolism (assessed by comparing 2004 and 2005 rates with previous measurements) was the result of the storm frequency and timing and the size of the spring macroalgal bloom. Changes in local climate can have substantial impacts on stream ecosystem metabolism rates and ultimately influence the carbon source and sink properties of these important ecosystems.     

Large-scale generation of highly enriched neural stem-cell-derived oligodendroglial cultures: maturation-dependent differences in insulin-like growth factor-mediated signal transduction

Multipotent neural stem cells (NSCs) are competent for commitment to the oligodendrocyte (OL) lineage both in vitro and in vivo. We exploited this property to develop a rat neurospheres (NS)/oligospheres (OS)-based culture system to generate large numbers of highly enriched late OL progenitors (preOLs) and mature OLs (MatOLs). CNS neuroblastoma cell line B104-derived conditioned medium promoted the generation of nearly pure populations of preOLs from dissociated OS. The subsequent culture of preOLs with ciliary neurotrophic factor (CNTF) and 3,3',5'-triiodo-L-thyronine (T(3)) generated nearly pure populations of MatOLs. OL lineage specificity was confirmed by immunocytochemistry, quantitative RT-PCR and gene expression profiling, which demonstrated large differences between preOLs and MatOLs. The insulin-like growth factors (IGFs) are potent neuro-protective agents required for OL survival. We used this system to systematically define maturation-dependent changes in IGF signaling during the course of OL differentiation. The IGF-I and insulin receptors, insulin receptor substrate-1 (IRS-1) and IRS-2, protein kinase B (PKB)/Akt and Janus kinase (JNK) were expressed at higher levels in NS and preOLs compared with OS and MatOLs. Erk expression increased markedly from NS to OS, decreased only partially upon commitment to preOLs, and, in MatOLs, returned to a low level similar to NS. IGF activation of the generally proliferative Erk pathway was gradually acquired during NSC differentiation, whereas IGF activation of the generally pro-survival, anti-apoptotic PI3K/PKB pathway was consistently robust at each developmental stage.     

Nightmares, life stress, and anxiety: An examination of tension reduction.

Do nightmares increase or decrease anxiety? Theoretical views of nightmares suggest that nightmares play out stressful events, decathecting their energy. A more pragmatic view suggests that nightmares that result in waking distress add to the burden of anxiety. The current study investigates whether negative life events are associated with an increase or decrease in anxiety attributable to nightmares in 624 adolescents aged between 12 and 19. The results indicate no support for a tension reduction hypothesis. There seems no relief from anxiety if a person reports nightmares, and the stronger the distress of waking (from nightmares), the more likely a person is to report anxiety, controlling for life events and the distress associated with life events. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Introduction of xylem differentiation in lactuca by ethylene

Evidence was obtained to support the hypothesis that ethylene is involved in xylem differentiation in primary pith explants of Lactuca sativa L. cv Romaine cultured in vitro. Xylem elements differentiated when explants were supplied indole-3-acetic acid (IAA) in combination with either the ethylene biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the ethylene-releasing agent 2-chloroethylphosphonic acid (CEPA), or kinetin. In contrast, no xylem elements differentiated in the presence of IAA, kinetin, ACC, or CEPA alone, or when kinetin was supplied together with ACC or CEPA. These results show that ethylene will substitute qualitatively for cytokinin during auxin-induced xylogenesis, and suggest that both ethylene and auxin are required for xylem differentiation in Lactuca.     

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2–4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5–7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTP-bound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs),² Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8–12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-containing RhoGEFs are a recently described family of GEFs. Currently, there are three members of this family, PDZ-RhoGEF, LARG, and p115RhoGEF (13–15), in which the RGS domains function as a heterotrimeric GTPase-activating domain (13, 15, 16). The RGS family of RhoGEFs has been shown to regulate Rho during several processes including cytoskeletal rearrangements, cell adhesion, and cancer progression (17–21).There is significant interplay between the activity of small GTPases and signaling derived from fatty acid metabolism (22–28). Linoleic acid, which is metabolized to arachidonic acid, is an n-6 polyunsaturated fatty acid that is present at high levels in most western diets (29). In animal models, diets high in n-6 polyunsaturated fatty acids have been shown to enhance tumor progression and metastasis (30, 31). Additionally, arachidonic acid is stored in cell membranes and is made available by phospholipases under conditions of increased inflammatory response (32). Arachidonic acid is further metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases to yield bioactive products that have myriad effects on cells, and altered metabolism of arachidonic acid by COX, LOX, and P450 has been implicated in cancer progression (31, 33–36).We have studied mechanisms of cell adhesion using the MDA-MB-435 cells as a model of a highly metastatic human cancer cell line (37). These cells have been extensively studied for their ability to recapitulate the metastatic cascade in vivo and in vitro, although recent work indicates that the cells currently in use are most likely a human melanoma line (38). We initially observed that arachidonic acid (AA) enhanced adhesion of MDA-MB-435 cells to type IV collagen through specific integrin-mediated pathways (37). Exogenous AA led to the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 and the phosphorylation of heat shock protein 27 (HSP27) via a p38 MAPK-dependent process (39). Inhibition of p38 MAPK activation blocked cell adhesion as did function-blocking antibodies specific for subunits of the collagen receptor (40). More recently, we identified the key metabolite of AA (15-(S)- hydroxyeicosatetraenoic acid) and the upstream kinases (TAK1 and MKK6) that are responsible for activation of p38 MAPK in this system (41).In this study we investigated the role of Rho activation in the MDA-MB-435 cells after exposure to arachidonic acid. Several aspects of the regulation of Rho signaling in these cells provide insights into the cross-talk between important signaling pathways.     

Probing the substrate specificities of human PHOSPHO1 and PHOSPHO2

PHOSPHO1, a phosphoethanolamine/phosphocholine phosphatase, is upregulated in mineralising cells and is thought to be involved in the generation of inorganic phosphate for bone mineralisation. PHOSPHO2 is a putative phosphatase sharing 42% sequence identity with PHOSPHO1. Both proteins contain three catalytic motifs, conserved within the haloacid dehalogenase superfamily. Mutation of Asp32 and Asp203, key residues within two motifs, abolish PHOSPHO1 activity and confirm it as a member of this superfamily. We also show that Asp43 and Asp123, residues that line the substrate-binding site in our PHOSPHO1 model, are important for substrate hydrolysis. Further comparative modelling reveals that the active sites of PHOSPHO1 and PHOSPHO2 are very similar, but surprisingly, recombinant PHOSPHO2 hydrolyses phosphoethanolamine and phosphocholine relatively poorly. Instead, PHOSPHO2 shows high specific activity toward pyridoxal-5-phosphate (V(max) of 633 nmol min(-1) mg(-1) and K(m) of 45.5 microM). Models of PHOSPHO2 and PHOSPHO1 suggest subtle differences in the charge distributions around the putative substrate entry site and in the location of potential H-bond donors.     

Structural study of the sugar chains of human platelet thrombospondin

The asparagine-linked sugar chains of human platelet thrombospondin were released as oligosaccharides by hydrazinolysis. About 12 mol of sugar chains was released from one thrombospondin molecule. This was converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation, and separated into one neutral and four acidic fractions by paper electrophoresis. More than 90% of the oligosaccharides were recovered in the acidic fraction. The acidic oligosaccharides were mostly converted to neutral oligosaccharides by sialidase treatment, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that the thrombospondin contains mono-, bi-, tri-, and tetraantennary complex-type sugar chains in addition to a small amount of high-mannose type. Approximately 70% of the complex-type sugar chains was fucosylated at asparagine-linked N-acetylglucosamine residue and 19% of the biantennary complex-type sugar chains was bisected.     

Homozygous paracentric inversion 12 in a mentally retarded boy: a case report and review of the literature

Summary A mentally retarded male was found to be homozygous for a paracentric inversion of the long arm of chromosome 12(inv(12)(q21.1q23.2)). His parents, who are first cousins, and his phenotypically normal younger brother are inversion heterozygotes. Homozygous structural rearrangements are discussed and cases of paracentric inversions, including a further nine previously unpublished, are reviewed.     

Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study

Nisin is a positively charged antibacterial peptide which binds to the negatively charged membranes of Gram-positive bacteria. The initial interaction of the peptide with model membranes of neutral (phosphatidylcholine) and negatively charged (phosphatidylcholine/phosphatidylglycerol) model lipid membranes was studied using nonperturbing solid state magic angle spinning (MAS) (31)P NMR and (2)H wide-line NMR. In the presence of nisin, the coexistence of two bilayer lipid environments was observed both in charged and in neutral membranes. One lipid environment was found to be associated with lipid directly interacting with nisin and one with noninteracting lipid. Solid state (31)P MAS NMR results show that the acidic membrane lipid component partitions preferentially into the nisin-associated environment. Deuterium NMR ((2)H NMR) of the selectively headgroup-labeled acidic lipid provides further evidence of a strong interaction between the charged lipid component and the peptide. The segregation of acidic lipid into the nisin-bound environment was quantified from (2)H NMR measurements of selectively headgroup-deuterated neutral lipid. It is suggested that the observed lipid partitioning in the presence of nisin is driven, at least initially, by electrostatic interactions. (2)H NMR measurements from chain-perdeuterated neutral lipids indicate that nisin perturbs the hydrophobic region of both charged and neutral bilayers.