Effects of juvenile hormone I and the anti-juvenile hormone fluoromevalono-lactone on development and juvenile hormone esterase activity in post-feeding last-stadium larvae of Trichoplusia ni (Hübner)

Treatment of post-feeding (early day 3; wandering phase) last-stadium larvae of the cabbage looper, Trichoplusia ni, with the anti-juvenile hormone, fluoromevalonolactone, prevented the normal ecdysis to the pupa. It caused the formation of larval-pupal intermediates, a dose-dependent delay in the time of tanning, and a decrease in juvenile hormone esterase activity at the time of the prepupal juvenile hormone esterase peak. Fluoromevalonolactone was inactive as juvenile hormone esterase inhibitor in vitro. Conversely, juvenile hormone I accelerated the time of tanning, induced the early appearance of juvenile hormone esterase activity, and prevented adult eclosion. Although most of the larvae that were treated with fluoromevalonolactone immediately after the prepupal burst of juvenile hormone (late on day 3; post-spinning phase) still became larval-pupal intermediates, the time of tanning and juvenile hormone esterase activity were close to normal. Topical treatment of day-3 larvae with radiolabelled juvenile hormone I resulted in the rapid appearance and decline of radiolabelled juvenile hormone I in the haemolymph which was associated with the increased production of juvenile hormone I acid and the induced appearance of juvenile hormone esterase activity. Thus, in post-feeding last-stadium larvae of T. ni, juvenile hormone seems to be necessary for the proper formation of the pupa. Juvenile hormone is also involved in determining the time of pupation, and it appears to induce its own degradation.     

Specific expression of proteins and phosphoproteins in 3T3 T mesenchymal stem cells at distinct growth arrest and differentiation states

Abstract. Murine mesenchymal stem cells can be induced to arrest their growth at a series of growth and differentiation states in the G₁ phase of the cell cycle. These include the predifferentiation arrest state (G_D) at which the integrated control of proliferation and differentiation is mediated, the growth factor/serum deficiency arrest state (G_S), and the nutrient deficiency arrest state (G_N). Cells at states of reversible nonterminal differentiation (G_D?) and irreversible terminal differentiation (TD) can also be isolated. In this paper we have employed 1- and 2-dimensional (D) gel electrophoresis to evaluate changes in specific proteins that occur during the various growth and differentiation states of 3T3 T mesenchymal stem cells. The protein composition of membrane, microsome and cytosol preparations of cells arrested at G_D, G_S and G_N states was determined by 2-D gel electrophoresis. More than 50 distinct polypeptides could be identified for each arrest state in gels analysed by a silver staining procedure or by autoradiography following [³⁵S]-methionine labelling. A second series of studies established that a more limited number of differences could be identified if phosphoproteins were analysed by 1-D gel electrophoresis in cells at the G_S, G_D, G_D?. and TD states. These results established that one distinct 37 kD phosphoprotein is present in all growth arrested cells and that two distinct differentiation-associated phosphoproteins with molecular weights of 29 kD and 72 kD are present in cells at the G_D? and TD states. Thus, the composition of proteins and phosphoproteins in mesenchymal stem cells serves to characterize different states of growth arrest and differentiation. The identification of differential protein expression provides an opportunity to test their functional role in growth and differentiation control.     

Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca²⁺]_cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca²⁺]_cyt were observed in response to glutamic acid (Glu), ATP, and aluminum (Al³⁺). Each chemical induced a [Ca²⁺]_cyt signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca²⁺]_cyt increases that were similar among the different root zones, whereas Al³⁺ evoked [Ca²⁺]_cyt transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al³⁺-induced [Ca²⁺]_cyt increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca²⁺]_cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca²⁺]_cyt signature resulting from Al³⁺ treatment originated mostly from cortical cells located at 300 to 500 μ m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca²⁺ channel blockers, elicited [Ca²⁺]_cyt responses similar to those induced by Al³⁺. The trivalent ion-induced [Ca²⁺]_cyt signatures in roots of an Al³⁺-resistant and an Al³⁺-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca²⁺]_cyt changes we report here may not be tightly linked to Al³⁺ toxicity but rather to a general response to trivalent cations.The role of calcium ions (Ca²⁺) as a ubiquitous cellular messenger in animal and plant cells is well established (Berridge et al., 2000; Sanders et al., 2002; Ng and McAinsh, 2003). Cellular signal transduction pathways are elicited as a result of fluctuations of free Ca²⁺ in the cytoplasm ([Ca²⁺]_cyt) in response to external and intracellular signals. These changes in [Ca²⁺]_cyt influence numerous cellular processes, including vesicle trafficking, cell metabolism, cell proliferation and elongation, stomatal opening and closure, seed and pollen grain germination, fertilization, ion transport, and cytoskeletal organization (Hepler, 2005). [Ca²⁺]_cyt fluctuations occur because cells have a Ca²⁺ signaling “toolkit” (Berridge et al., 2000) composed of on/off switches and a multitude of Ca²⁺-binding proteins. The on switches depend on membrane-localized Ca²⁺ channels that control the entry of Ca²⁺ into the cytosol (Piñeros and Tester, 1995, 1997; Thion et al., 1998; Kiegle et al., 2000a; White et al., 2000; Demidchik et al., 2002; Miedema et al., 2008). On the other hand, the off switches consist of a family of Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers in the plasma membrane or endomembrane that remove Ca²⁺ from the cytosol, bringing the [Ca²⁺]_cyt down to the initial resting level (Lee et al., 2007; Li et al., 2008).The numerous cellular processes regulated by Ca²⁺ have led investigators to ask how specificity in Ca²⁺ signaling is maintained. It has been proposed that specificity in Ca²⁺ signaling is achieved because a particular stimulus elicits a distinct Ca²⁺ signature, which is defined by the timing, magnitude, and frequency of [Ca²⁺]_cyt changes. For instance, tip-growing plant cells such as root hairs and pollen tubes exhibit oscillatory elevations in [Ca²⁺]_cyt that partly mirror the oscillatory nature of growth in these cell types (Cárdenas et al., 2008; Monshausen et al., 2008). Another example is nuclear Ca²⁺ spiking in root hairs of legumes exposed to NOD factors (Oldroyd and Downie, 2006; Peiter et al., 2007). Recently, it was shown that mechanical forces applied to an Arabidopsis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca²⁺]_cyt response (Monshausen et al., 2009). Translating the Ca²⁺ signature into a defined cellular response is governed by a number of Ca²⁺-binding proteins such as calreticulin that act as [Ca²⁺]_cyt buffers, which shape both the amplitude and duration of the Ca²⁺ signal or Ca²⁺ sensors such as calmodulin that impact other downstream cellular effectors (Berridge et al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca²⁺ signaling mechanisms in plants has been driven in large part by our ability to monitor dynamic changes in [Ca²⁺]_cyt in the cell. Such measurements have been conducted using Ca²⁺-sensitive fluorescent indicator dyes (e.g. Indo and Fura), the luminescent protein aequorin (Knight et al., 1991, 1996; Legué et al., 1997; Wymer et al., 1997; Cárdenas et al., 2008), and more recently the yellow cameleon (YC) Ca²⁺ sensor, a chimeric protein that relies on fluorescence resonance energy transfer (FRET) as an indicator of [Ca²⁺]_cyt changes in the cell (Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006; Tang et al., 2007; Haruta et al., 2008). The YC reporter is composed of cyan fluorescent protein (CFP), the C terminus of calmodulin (CaM), a Gly-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and a yellow fluorescent protein (YFP; Miyawaki et al., 1997, 1999). The increased interaction between M13 and CaM upon binding of Ca²⁺ to CaM triggers a conformational change in the protein that brings the CFP and YFP in close proximity, resulting in enhanced FRET efficiency between the two fluorophores (Miyawaki, 2003). Thus, changes in FRET efficiency between CFP and YFP in the cameleon reporter are correlated with changes in [Ca²⁺]_cyt.Since it was first introduced, improved versions of the cameleon reporter have been selected to more accurately report [Ca²⁺]_cyt levels in the cell. For instance, the YC3.60 version was selected because of its resistance to cytoplasmic acidification and its higher dynamic range compared with the earlier cameleons. The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al., 2004). Recently, the utility of YC3.60 for monitoring [Ca²⁺]_cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al., 2007, 2008, 2009; Haruta et al., 2008; Iwano et al., 2009). Here, we further evaluated YC3.60 as a [Ca²⁺]_cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging. Unlike the direct ratiometric measurement of cpVenus and CFP reported in previous studies using YC3.60-expressing plants (Monshausen et al., 2008, 2009), the sensitized FRET approach we describe here involves the use of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-through and background signals from the FRET specimen (van Rheenen et al., 2004; Feige et al., 2005).For this study, we focused on monitoring [Ca²⁺]_cyt changes in Arabidopsis seedling roots after aluminum (Al³⁺) exposure. Although Ca²⁺ signaling has long been implicated in mediating Al³⁺ responses in plants (Rengel and Zhang, 2003), the [Ca²⁺]_cyt changes evoked by Al³⁺ reported in the literature have been inconsistent, and as such, the significance of these [Ca²⁺]_cyt responses to mechanisms of Al³⁺ toxicity are not very clear. For instance, some studies reported that Al³⁺ caused a decrease in [Ca²⁺]_cyt in plants (Jones et al., 1998b; Kawano et al., 2004), and others demonstrated elevated [Ca²⁺]_cyt upon Al³⁺ treatment (Nichol and Oliveira, 1995; Lindberg and Strid, 1997; Jones et al., 1998a; Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004).Here, we report that Arabidopsis roots expressing the YC3.60 reporter exhibited transient elevations in [Ca²⁺]_cyt within seconds of Al³⁺ exposure. The general pattern of [Ca²⁺]_cyt changes observed after Al³⁺ treatment were distinct from those elicited by ATP or Glu, reinforcing the concept of specificity in [Ca²⁺]_cyt signaling. We also observed root zone-dependent variations in the [Ca²⁺]_cyt signatures evoked by Al³⁺ in regard to the shape, duration, and timing of the [Ca²⁺]_cyt response. Other trivalent ions such as lanthanum (La³⁺) and gadolinium (Ga³⁺), which have been widely used as Ca²⁺ channel blockers (Monshausen et al., 2009), also induced a rapid rise in [Ca²⁺]_cyt in root cells that were similar to those elicited by Al³⁺. Al³⁺, La³⁺, and Gd³⁺ elicited similar [Ca²⁺]_cyt signatures in the Al³⁺-tolerant mutant alr104 (Larsen et al., 1998) and the Al³⁺-sensitive mutant als3-1 (Larsen et al., 2005), indicating that the early [Ca²⁺]_cyt increases we report here may not be tightly linked to mechanisms of Al³⁺ toxicity but rather to a general trivalent cation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis roots expressing YC3.60 provides a robust method for documenting [Ca²⁺]_cyt signatures in different root developmental zones that should be useful for future studies on Ca²⁺ signaling mechanisms in plants.     

Evaluation of systems for collection of porcine zygotes for DNA microinjection and transfer

Crossbred gilts and sows (n=116) were used for the collection of 1-cell zygotes for DNA microinjection and transfer. Retrospectively, estrus synchronization and superovulation schemes were evaluated to assess practicality for zygote collection. Four synchronization and superovulation procedures were used: 1) sows were observed for natural estrous behavior; 1000 IU human chorionic gonadotrophin (hCG) was administered at the onset of estrus (NAT); 2) cyclic gilts were synchronized with 17.6 mg altrenogest (ALT)/day for 15 to 19 days followed by superovulation with 1500 IU pregnant mares serum gonadotropin (PMSG) and 500 IU hCG (LALT); 3) gilts between 11 and 16 days of the estrous cycle received 17.6 mg ALT for 5 to 9 days and PMSG and hCG were used to induce superovulation (SALT); and 4) precocious ovulation was induced in prepubertal gilts with PMSG and hCG (PRE). A total of 505 DNA microinjected embryos transferred into 17 recipients produced 7 litters and 50 piglets, of which 8 were transgenic. The NAT sows had less (P < 0.05) ovarian activity than gilts synchronized and superovulated by all the other procedures. Synchronization treatments with PMSG did not differ (P > 0.05) in the number of corpora hemorrhagica or unovulated follicles, but SALT and PRE treaments had higher ovulation rates than LALT (24.7 +/- 2.9, 24.3 +/- 1.8 vs 11.6 +/- 2.7 ovulations; X +/- SEM). The SALT and PRE treatments yielded 12.3 +/- 2.6 and 17.7 +/- 1.7 zygotes. Successful transgenesis was accomplished with SALT and PRE procedures for estrus synchronization and superovulation.     

The Toxicity of Sulphur Dioxide towards Certain Lactic Acid Bacteria from Fermented Apple Juice

This paper describes a method for testing the effect of various concentrations of SO₂ on lactic acid bacteria from ciders. The media and methods were devised to minimize loss of SO₂ due to oxidation or binding with carbonyl compounds. Exposure of laboratory or freshly isolated strains to various concentrations of free SO₂ at pH 4·0 did not readily kill them even at high concentrations of free SO₂ ( c. 150 p/m or 0·97 p/m molecular SO₂) yet they were suppressed at low concentrations ( c. 5 p/m or 0·032 p/m molecular SO₂). Reducing the pH to 3·4 reaffirmed how much more effective SO₂ is against lactic acid bacteria at lower pH levels because more is present as molecular SO₂. As a result of this the idea of quoting SO₂ values as p/m molecular SO₂ is advocated. Addition of hydrogen peroxide or acetaldehyde to a test system containing 142 p/m free SO₂ showed that they had a similar effect in nullifying its antimicrobial properties and allowing the test bacteria to grow. There was no indication that acetaldehyde bisulphite was toxic to the test bacteria.     

Estimating Ecosystem Nitrogen Addition by a Leguminous Tree: A Mass Balance Approach Using a Woody Encroachment Chronosequence

Difficulty in quantifying rates of biological N fixation (BNF), especially over long time scales, remains a major impediment to defining N budgets in many ecosystems. To estimate N additions from BNF, we applied a tree-scale N mass balance approach to a well-characterized chronosequence of woody legume (Prosopis glandulosa) encroachment into subtropical grasslands. We defined spatially discrete single Prosopis clusters (aged 28–99 years), and for each calculated BNF as the residual of: soil N (0–30 cm), above- and below-ground biomass N, wet and dry atmospheric N deposition, N trace gas and N₂ loss, leaching loss, and baseline grassland soil N at time of establishment. Contemporary BNF for upland savanna woodland was estimated at 10.9 ± 1.8 kg N ha^?1 y^?1, equal to a total of 249 ± 60 kg N ha^?1 over about 130 years of encroachment at the site. Though these BNF values are lower than previous estimates for P. glandulosa, this likely reflects lower plant density as well as low water availability at this site. Uncertainty in soil and biomass parameters affected BNF estimates by 6–11%, with additional sensitivity of up to 18% to uncertainty in other scaling parameters. Differential N deposition (higher rates of dry N deposition to Prosopis canopies versus open grasslands) did not explain N accrual beneath trees; iterations that represented this scenario reduced estimated BNF estimates by a maximum of 1.5 kg N ha^?1 y^?1. We conclude that in this relatively well-constrained system, small-scale mass balance provides a reasonable method of estimating BNF and could provide an opportunity to cross-calibrate alternative estimation approaches.     

ConA的抗着床效应

本文用凝集素为探针,探索糖复合物在胚泡着床中的作用,报道了与甘露糖苷有专一结合的伴刀豆凝集素(ConA)有明显的抗小鼠胚泡着床作用。妊娠4d的小鼠,每只子宫角中注入Con A 25μg,22只子宫角中只有4只子宫角有胚泡着床,着床率为18.2％,与生理盐水对照组的着床率87.5％相比有明显差异。将相同剂量的Con A先与0.4mol/L α-甲基-D-甘露糖苷温育1—2h后再注入子宫,20只子宫角中有15只子宫角有胚泡着床,着床率提高到75％。用辣根过氧化物酶直接标记法证明,着床前子宫内膜细胞表面有Con A受体存在,并随着妊娠天数而增加,尤其是间质细胞,发情期时时为阴性反应,到着床期蜕膜细胞膜表面呈现出大量Con A受体。提示精复合物在着床中的重要作用。与甘露糖苷同样专一结合的,但寡糖结构专一性与Con A不同的豌豆凝集素注入子宫则无抗着床效应,着床率为85.7％。由此可以推测,N-连接的包含二个未被取代的或只在C-2位被取代的α-甘露糖苷的寡糖参于胚泡与子宫内膜相互作用的着床过程。     

Why experiment with success? Opportunities and risks in applying assessment and adaptive management to the Emiquon floodplain restoration project

The Nature Conservancy’s wetland restoration at the Emiquon Preserve has been a success to date, but there are warning signs of undesirable change if left unmanaged. A water control structure built in 2016 will increase management capabilities, but periodic connection to the river, which has experienced human alterations typical of rivers in eastern North America and Europe, also introduces risks. The Conservancy’s planning process has identified (1) management targets (e.g., diverse native fish populations); (2) Key Ecological Attributes (KEAs) that maintain the targets (e.g., relatively deep over-wintering habitats for fishes); (3) measurable indicators for the KEAs (e.g., depth in winter); and (4) desirable ranges for the indicators (e.g., 10% of the aquatic area has depths of 2–3 m and dissolved oxygen levels of 4–6 mg/l). Assessments and experiments completed to date have focused on documenting the restoration, evaluating effects of the record flood of 2013, and predicting outcomes of management actions. Simulation models of hydrology, hydraulics, and vegetation response developed during the planning process allayed some concerns of stakeholders, but not all outcomes are predictable from either current theory or management experience. Therefore, each action can be considered not only as an adaptive management experiment focused on sustaining targets, but also contributing to ecological theory and restoration practice on a broader scale.