Genetic selection for modulators of the MAP kinase and beta-catenin growth-control pathways in mammalian cells

Transdominant genetic selections can yield protein fragment and peptide modulators of specific biochemical pathways. In yeast, such screens have been highly successful in targeting the MAP (mitogen-activated protein) kinase growth-control pathway. We performed a similar type of selection aimed at recovery of modulators of the mammalian MAP kinase cascade. Two pathway activators were identified, fragments of the TrkB and Raf-1 kinases. In a second selection directed at the beta-catenin growth-control pathway, three different clones encoding cadherin fragments were recovered. In neither selection were peptide inhibitors observed. We conclude that some transdominant selections in mammalian cells can readily yield high-penetrance protein fragments, but may be less amenable to isolation of peptide inhibitors.     

Loss of oxidized and chlorinated bases in DNA treated with reactive oxygen species: implications for assessment of oxidative damage in vivo

Oxidative damage to DNA has been reported to occur in a wide variety of disease states. The most widely used "marker" for oxidative DNA damage is 8-hydroxyguanine. However, the use of only one marker has limitations. Exposure of calf thymus DNA to an .OH-generating system (CuCl(2), ascorbate, H(2)O(2)) or to hypochlorous acid (HOCl), led to the extensive production of multiple oxidized or chlorinated DNA base products, as measured by gas chromatography-mass spectrometry. The addition of peroxynitrite (ONOO(-)) (<200 microM) or SIN-1 (1mM) to oxidized DNA led to the extensive loss of 8-hydroxyguanine, 5-hydroxycytosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2-hydroxyadenine, 8-hydroxyadenine, and 4,6-diamino-5-formamidopyrimidine were lost at higher ONOO(-) concentrations (>200 microM). Exposure of DNA to HOCl led to the generation of 5-Cl uracil and 8-Cl adenine and addition of ONOO(-) (<200 microM) or SIN-1 (1mM) led to an extensive loss of 8-Cl adenine and a small loss of 5-Cl uracil at higher concentrations (>500 microM). An .OH-generating system (CuCl(2)/ascorbate/H(2)O(2)) could also destroy these chlorinated species. Treatment of oxidized or chlorinated DNA with acidified nitrite (NO(2)(-), pH 3) led to substantial loss of various base lesions, in particular 8-OH guanine, 5-OH cytosine, thymine glycol, and 8-Cl adenine. Our data indicate the possibility that when ONOO(-), nitrite in regions of low pH or .OH are produced at sites of inflammation, levels of certain damaged DNA bases could represent an underestimate of ongoing DNA damage. This study emphasizes the need to examine more than one modified DNA base when assessing the role of reactive species in human disease.     

Presenilin-1 Dependent Neurogenesis Regulates Hippocampal Learning and Memory

Presenilin-1 (PS1), the catalytic core of the aspartyl protease γ-secretase, regulates adult neurogenesis. However, it is not clear whether the role of neurogenesis in hippocampal learning and memory is PS1-dependent, or whether PS1 loss of function in adult hippocampal neurogenesis can cause learning and memory deficits. Here we show that downregulation of PS1 in hippocampal neural progenitor cells causes progressive deficits in pattern separation and novelty exploration. New granule neurons expressing reduced PS1 levels exhibit decreased dendritic branching and dendritic spines. Further, they exhibit reduced survival. Lastly, we show that PS1 effect on neurogenesis is mediated via β-catenin phosphorylation and notch signaling. Together, these observations suggest that impairments in adult neurogenesis induce learning and memory deficits and may play a role in the cognitive deficits observed in Alzheimer’s disease.     

Reduced Fitness of Virulent Aphis glycines (Hemiptera: Aphididae) Biotypes May Influence the Longevity of Resistance Genes in Soybean

Sustainable use of insect resistance in crops require insect resistance management plans that may include a refuge to limit the spread of virulence to this resistance. However, without a loss of fitness associated with virulence, a refuge may not prevent virulence from becoming fixed within a population of parthenogenetically reproducing insects like aphids. Aphid-resistance in soybeans (i.e., Rag genes) prevent outbreaks of soybean aphid (Aphis glycines), yet four biotypes defined by their capacity to survive on aphid-resistant soybeans (e.g., biotype-2 survives on Rag1 soybean) are found in North America. Although fitness costs are reported for biotype-3 on aphid susceptible and Rag1 soybean, it is not clear if virulence to aphid resistance in general is associated with a decrease in fitness on aphid susceptible soybeans. In laboratory assays, we measured fitness costs for biotype 2, 3 and 4 on an aphid-susceptible soybean cultivar. In addition, we also observed negative cross-resistance for biotype-2 on Rag3, and biotype-3 on Rag1 soybean. We utilized a simple deterministic, single-locus, four compartment genetic model to account for the impact of these findings on the frequency of virulence alleles. When a refuge of aphid susceptible was included within this model, fitness costs and negative cross-resistance delayed the increase of virulence alleles when virulence was inherited recessively or additively. If virulence were inherited additively, fitness costs decreased the frequency of virulence. Combined, these results suggest that a refuge may prevent virulent A. glycines biotypes from overcoming Rag genes if this aphid-resistance were used commercially in North America.     

Ultrastructure of Wax-Producing Structures on the Integument of the Melaleuca Psyllid Boreioglycaspis melaleucae (Hemiptera: Psyllidae), with Honeydew Excretion Behavior in Males and Females

The melaleuca psyllid, Boreioglycaspis melaleucae (Hemiptera: Psyllidae), was introduced to Florida as a biological control agent against Melaleuca quinquenervia, an invasive evergreen tree that has invaded large areas of Florida Everglades. Colonies of B. melaleucae nymphs are normally covered by white waxy secretions, and nymphs of various instars produce long bundles of white waxy filaments extending laterally and posteriorly from their abdomen. Scanning electron microscopy of ‘naturally waxed’ and ‘dewaxed’ nymphs (cleaned from wax) revealed two types of wax pore plates located dorsally and laterally on the integument of posterior abdominal segments starting with the 4th segment. Type-1 wax pore plates, with raised rim, peripheral groove, slits and pits, produce long ribbons and filaments of waxy secretions that are wound together forming long wax bundles, whereas type-2 wax pore plates, with slits only, produce shorter wax curls. Additionally, in both nymphs and adult females, the circumanal ring contained ornate rows of wax pores that produce wax filaments covering their honeydew excretions. Video recordings with stereomicroscopy showed that adult females produce whitish honeydew balls, powerfully propelled away from their body, probably to get these sticky excretions away from their eggs and newly hatched nymphs. Adult males, however, produce clear droplets of honeydew immediately behind them, simply by bending the posterior end of the abdomen downward. The possible role(s) of waxy secretions by nymphs and adults of B. melaleucae in reducing contamination of their colonies with honeydew, among other possibilities, are discussed.     

Class XI Myosins Move Specific Organelles in Pollen Tubes and Are Required for Normal Fertility and Pollen Tube Growth in Arabidopsis

Pollen tube growth is an essential aspect of plant reproduction because it is the mechanism through which nonmotile sperm cells are delivered to ovules, thus allowing fertilization to occur. A pollen tube is a single cell that only grows at the tip, and this tip growth has been shown to depend on actin filaments. It is generally assumed that myosin-driven movements along these actin filaments are required to sustain the high growth rates of pollen tubes. We tested this conjecture by examining seed set, pollen fitness, and pollen tube growth for knockout mutants of five of the six myosin XI genes expressed in pollen of Arabidopsis (Arabidopsis thaliana). Single mutants had little or no reduction in overall fertility, whereas double mutants of highly similar pollen myosins had greater defects in pollen tube growth. In particular, myo11c1 myo11c2 pollen tubes grew more slowly than wild-type pollen tubes, which resulted in reduced fitness compared with the wild type and a drastic reduction in seed set. Golgi stack and peroxisome movements were also significantly reduced, and actin filaments were less organized in myo11c1 myo11c2 pollen tubes. Interestingly, the movement of yellow fluorescent protein-RabA4d-labeled vesicles and their accumulation at pollen tube tips were not affected in the myo11c1 myo11c2 double mutant, demonstrating functional specialization among myosin isoforms. We conclude that class XI myosins are required for organelle motility, actin organization, and optimal growth of pollen tubes.Pollen tubes play a crucial role in flowering plant reproduction. A pollen tube is the vegetative cell of the male gametophyte. It undergoes rapid polarized growth in order to transport the two nonmotile sperm cells to an ovule. This rapid growth is supported by the constant delivery of secretory vesicles to the pollen tube tip, where they fuse with the plasma membrane to enlarge the cell (Bove et al., 2008; Bou Daher and Geitmann, 2011; Chebli et al., 2013). This vesicle delivery is assumed to be driven by the rapid movement of organelles and cytosol throughout the cell, a process that is commonly referred to as cytoplasmic streaming (Shimmen, 2007). Cytoplasmic streaming in angiosperm pollen tubes forms a reverse fountain: organelles moving toward the tip travel along the cell membrane, while organelles moving away from the tip travel through the center of the tube (Heslop-Harrison and Heslop-Harrison, 1990; Derksen et al., 2002). Drug treatments revealed that pollen tube cytoplasmic streaming and tip growth depend on actin filaments (Franke et al., 1972; Mascarenhas and Lafountain, 1972; Heslop-Harrison and Heslop-Harrison, 1989; Parton et al., 2001; Vidali et al., 2001). Curiously, very low concentrations of actin polymerization inhibitors can prevent growth without completely stopping cytoplasmic streaming, indicating that cytoplasmic streaming is not sufficient for pollen tube growth (Vidali et al., 2001). At the same time, however, drug treatments have not been able to specifically inhibit cytoplasmic streaming; thus, it is unknown whether cytoplasmic streaming is necessary for pollen tube growth.Myosins are actin-based motor proteins that actively transport organelles throughout the cell and are responsible for cytoplasmic streaming in plants (Shimmen, 2007; Sparkes, 2011; Madison and Nebenführ, 2013). Myosins can be grouped into at least 30 different classes based on amino acid sequence similarity of the motor domain, of which only class VIII and class XI myosins are found in plants (Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Class VIII and class XI myosins have similar domain architecture. The N-terminal motor domain binds actin and hydrolyzes ATP (Tominaga et al., 2003) and is often preceded by an SH3-like (for sarcoma homology3) domain of unknown function. The neck domain, containing IQ (Ile-Gln) motifs, acts as a lever arm and is bound by calmodulin-like proteins that mediate calcium regulation of motor activity (Kinkema and Schiefelbein, 1994; Yokota et al., 1999; Tominaga et al., 2012). The coiled-coil domain facilitates dimerization (Li and Nebenführ, 2008), and the globular tail functions as the cargo-binding domain (Li and Nebenführ, 2007). Class VIII myosins also contain an N-terminal extension, MyTH8 (for myosin tail homology8; Mühlhausen and Kollmar, 2013), and class XI myosins contain a dilute domain in the C-terminal globular tail (Kinkema and Schiefelbein, 1994; Odronitz and Kollmar, 2007; Sebé-Pedrós et al., 2014). Recently, Mühlhausen and Kollmar (2013) proposed a new nomenclature for plant myosins based on a comprehensive phylogenetic analysis of all known plant myosins that clearly identifies paralogs and makes interspecies comparisons easier (Madison and Nebenführ, 2013).The localization of class VIII myosins, as determined by immunolocalization and the expression of fluorescently labeled full-length or tail constructs, has implicated these myosins in cell-to-cell communication, cell division, and endocytosis in angiosperms and moss (Reichelt et al., 1999; Van Damme et al., 2004; Avisar et al., 2008; Golomb et al., 2008; Sattarzadeh et al., 2008; Yuan et al., 2011; Haraguchi et al., 2014; Wu and Bezanilla, 2014). On the other hand, class XI myosin mutants have been studied extensively in Arabidopsis (Arabidopsis thaliana), which revealed roles for class XI myosins in cell expansion and organelle motility (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Very few studies have examined the reproductive tissues of class XI myosin mutants. In rice (Oryza sativa), one myosin XI was shown to be required for normal pollen development under short-day conditions (Jiang et al., 2007). In Arabidopsis, class XI myosins are required for stigmatic papillae elongation, which is necessary for normal fertility (Ojangu et al., 2012). Even though pollen tubes of myosin XI mutants have not been examined, the tip growth of another tip-growing plant cell has been thoroughly examined in myosin mutants. Root hairs are tubular outgrowths of root epidermal cells that function to increase the surface area of the root for water and nutrient uptake. Two myosin XI mutants have shorter root hairs, of which the myo11e1 (xik; myosin XI K) mutation has been shown to be associated with a slower root hair growth rate and reduced actin dynamics compared with the wild type (Ojangu et al., 2007; Peremyslov et al., 2008; Park and Nebenführ, 2013). Higher order mutants have a further reduction in root hair growth and have altered actin organization (Prokhnevsky et al., 2008; Peremyslov et al., 2010). Disruption of actin organization was also observed in myosin XI mutants of the moss Physcomitrella patens (Vidali et al., 2010), where these motors appear to coordinate the formation of actin filaments in the apical dome of the tip-growing protonemal cells (Furt et al., 2013). Interestingly, organelle movements in P. patens are much slower than in angiosperms and do not seem to depend on myosin motors (Furt et al., 2012).The function of myosins in pollen tubes is currently not known, although it is generally assumed that they are responsible for the prominent cytoplasmic streaming observed in these cells by associating with organelle surfaces (Kohno and Shimmen, 1988; Shimmen, 2007). Myosin from lily (Lilium longiflorum) pollen tubes was isolated biochemically and shown to move actin filaments with a speed of about 8 µm s⁻¹ (Yokota and Shimmen, 1994) in a calcium-dependent manner (Yokota et al., 1999). Antibodies against this myosin labeled small structures in both the tip region and along the shank (Yokota et al., 1995), consistent with the proposed role of this motor in moving secretory vesicles to the apex.In Arabidopsis, six of 13 myosin XI genes are highly expressed in pollen: Myo11A1 (XIA), Myo11A2 (XID), Myo11B1 (XIB), Myo11C1 (XIC), Myo11C2 (XIE), and Myo11D (XIJ; Peremyslov et al., 2011; Sparkes, 2011). The original gene names (Reddy and Day, 2001) are given in parentheses. Myo11D is the only short-tailed myosin XI in Arabidopsis (Mühlhausen and Kollmar, 2013) and lacks the typical myosin XI globular tail involved in cargo binding (Li and Nebenführ, 2007). The remaining genes have the same domain architecture as the conventional class XI myosins that have been shown to be involved in the elongation of trichomes, stigmatic papillae, and root hairs (Ojangu et al., 2007, 2012; Peremyslov et al., 2008, 2010; Prokhnevsky et al., 2008; Park and Nebenführ, 2013). Therefore, we predicted that these five pollen-expressed, conventional class XI myosins are required for the rapid elongation of pollen tubes. In this study, we examined transfer DNA (T-DNA) insertion mutants of Myo11A1, Myo11A2, Myo11B1, Myo11C1, and Myo11C2 for defects in fertility and pollen tube growth. Organelle motility and actin organization were also examined in myo11c1 myo11c2 pollen tubes.     

Behavioural Effects of Tourism on Oceanic Common Dolphins,Delphinus sp., in New Zealand: The Effects of Markov Analysis Variations and Current Tour Operator Compliance with Regulations

Common dolphins, Delphinus sp., are one of the marine mammal species tourism operations in New Zealand focus on. While effects of cetacean-watching activities have previously been examined in coastal regions in New Zealand, this study is the first to investigate effects of commercial tourism and recreational vessels on common dolphins in an open oceanic habitat. Observations from both an independent research vessel and aboard commercial tour vessels operating off the central and east coast Bay of Plenty, North Island, New Zealand were used to assess dolphin behaviour and record the level of compliance by permitted commercial tour operators and private recreational vessels with New Zealand regulations. Dolphin behaviour was assessed using two different approaches to Markov chain analysis in order to examine variation of responses of dolphins to vessels. Results showed that, regardless of the variance in Markov methods, dolphin foraging behaviour was significantly altered by boat interactions. Dolphins spent less time foraging during interactions and took significantly longer to return to foraging once disrupted by vessel presence. This research raises concerns about the potential disruption to feeding, a biologically critical behaviour. This may be particularly important in an open oceanic habitat, where prey resources are typically widely dispersed and unpredictable in abundance. Furthermore, because tourism in this region focuses on common dolphins transiting between adjacent coastal locations, the potential for cumulative effects could exacerbate the local effects demonstrated in this study. While the overall level of compliance by commercial operators was relatively high, non-compliance to the regulations was observed with time restriction, number or speed of vessels interacting with dolphins not being respected. Additionally, prohibited swimming with calves did occur. The effects shown in this study should be carefully considered within conservation management plans, in order to reduce the risk of detrimental effects on common dolphins within the region.     

Analysis of lipocyte viability after liposuction

Free fat grafts from liposuction aspirate can be used as donor material for soft-tissue augmentation. The purpose of this study was to attempt to identify a subpopulation of adipose cells within liposuction aspirate with the greatest viability and, it is hoped, a greater chance for increased survival after transplantation. Liposuction samples were obtained from 20 individuals (16 women, four men; age range, 27 to 49 years). These samples were then centrifuged at 50 g. At 2-minute intervals, specimens from three different areas (superficial, middle, deep) were obtained from each specimen. After collagenase degradation, the specimens were stained with trypan blue, and the number of viable cells were counted. The bottom (deepest) layer consistently contained the highest number of viable cells after centrifugation: 250 percent more viable cells when compared with the top layer (p < 0.0001) and 140 percent more viable cells when compared with the middle layer (p < 0.0002). Centrifugation beyond 2 minutes did not increase the number or proportion of viable adipocytes. When using aspirated fat from liposuction for soft-tissue augmentation, centrifugation for 2 minutes at 50 g will stratify the adipocytes, with more viable cells being found at the deepest layer. Using only this bottom portion of the fat layer for transplantation will yield a fat graft with a greater number of viable adipocytes, potentially improving fat graft survival and decreased fat graft resorption.     

Correlation of the kinetics of finger domain mutants in RB69 DNA polymerase with its structure

We have estimated pre-steady-state kinetic parameters for the addition of a single nucleotide residue by a set of RB69 DNA polymerase mutants in which four highly conserved residues in the fingers domain have been replaced by Ala. The relationship between the kinetic constants exhibited by the mutants and the structure of the ternary complex [Franklin, M., Wang, J., and Steitz T. (2001) Cell 105, 657-667] was consistent with the following sets of interactions between the conserved residues and oxygen atoms in the triphosphate portion of the incoming dNTP: (i) the epsilon-amino group of K560 contacts oxygen atoms of the alpha- and gamma-phosphates, (ii) the amide side chain of Asn 564 forms a hydrogen bond via a water molecule with the nonbridging oxygen of the beta-phosphate, and (iii) the epsilon-amino and delta-guanidino groups of K486 and R482, respectively, contact the nonbridging oxygens of the gamma-phosphate. We have also determined the pre-steady-state kinetic parameters for the addition of both dCTP and dCDP onto a 13/20mer primer/template with an exo(-) derivative of RB69 DNA polymerase and have shown that the deoxynucleoside diphosphate can be incorporated, in contrast to the behavior of the Klenow fragment which cannot use dCDP as a substrate. We have shown that, with RB69 DNA polymerase, in contrast to the Klenow fragment, there is no inhibition of the primer-extension reaction by incoming NTPs having either noncomplementary bases or ribo- instead of a deoxyribose moieties. This implies that the mode of recognition of incoming dNTPs and triggering of the conformational change, which is thought to occur prior to the chemical step, differs between these two enzymes.