The Ecological State of the Waddensea. An Assessment

Since the present quality state of the Wadden Sea is judged more discordantly than that of the North Sea, it is examined whether the Wadden Sea is a separate ecosystem. The nutrient load into the Wadden Sea and the trend of the load are assessed. Biological indicators of the ecological state of the Wadden Sea are examined with the result that there are signs of a bad as well as of a good state. This contradiction is assigned to the fact that quality standards are absent. Self-protection mechanisms of the Wadden Sea are discussed with respect to their responsibility for the relatively good state. A qualitative model is proposed to explain the long-term behaviour of the ecosystem.     

加拿大一枝黄花入侵的细胞学机制

对入侵植物加拿大一枝黄花(Solidago canadensis L.)和同属土著种一枝黄花(Solidago decurrens Lour.)的染色体计数,并对核型进行了分析.实验结果:加拿大一枝黄花染色体数目为2n=54,核型公式为k(2n)=6x=54＝46m 8sm(0-6SAT),核型类型为2A型;一枝黄花染色体数目为2n=18,核型公式为k(2n)=2x=18=16m 2sm(0-2SAT),核型类型为1A型.通过对一枝黄花属(Solidago L.)植物染色体数目的统计分析,判断该属的染色体基数为9.通过对多倍体基因表达导致植物适应进化的讨论得出:多倍体是入侵植物特征,可能是植物入侵的内在机制.     

Season‐long mating disruption of citrus leafminer,Phyllocnistis citrella Stainton,with an emulsified wax formulation of pheromone

The citrus leafminer, Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), is a major worldwide pest of citrus. Larval feeding by this insect facilitates proliferation of citrus bacterial canker, Xanthomonas axonopodis pv. citri. Herein, we describe a season‐long disruption trial of P. citrella with a newly developed, emulsified wax dispenser of pheromone (SPLAT‐CLM^TM). A formulation containing a 3 : 1 blend of (Z,Z,E)‐7,11,13‐hexadecatrienal:(Z,Z)‐7,11‐hexadecadienal at a 0.2% loading rate of active ingredient by weight and deployed twice per season (24 weeks total) at 490 g of formulation/ha caused season‐long disruption of male moth catch in pheromone traps as well as reduced leaf infestation. Analysis of pheromone release from dispensers by gas chromatography revealed that effective disruption of P. citrella occurred at a deployment rate of 126 μg of (Z,Z,E)‐7,11,13‐hexadecatrienal/ha/h. Direct observation of moth behaviour in the field suggested that disruption by this formulation occurred by a non‐competitive mechanism. A formulation of the 3 : 1 attractive blend at a 0.02% pheromone loading rate caused only 2–6 weeks of disruption per deployment and did not reduce leaf infestation during mid and end of the season evaluations. A formulation containing 0.2% of (Z,Z)‐7,11‐hexadecadienal alone and deployed at 490 g/ha caused 6–7 weeks of moth disruption to pheromone traps and did not prevent leaf infestation, while an identical formulation loaded with 0.02% (w/w) of (Z,Z)‐7,11‐hexadecadienal alone had no effect on P. citrella orientation to pheromone traps. The SPLAT formulation evaluated herein appears to be an excellent release device for (Z,Z,E)‐7,11,13‐hexadecatrienal given that approximately 100 days of steady release occurred following an initial brief (ca. 7 days) burst of higher release. The advantages of SPLAT as a formulation for P. citrella disruption include low cost of manufacturing, biodegradable and weather resistant characteristics, and flowability allowing machine application. Mating disruption should be an effective alternative to insecticides for management of P. citrella and may reduce the incidence of citrus canker.     

Actin depolymerizing factor stabilizes an existing state of F-actin and can change the tilt of F-actin subunits

Proteins in the actin depolymerizing factor (ADF)/cofilin family are essential for rapid F-actin turnover, and most depolymerize actin in a pH-dependent manner. Complexes of human and plant ADF with F-actin at different pH were examined using electron microscopy and a novel method of image analysis for helical filaments. Although ADF changes the mean twist of actin, we show that it does this by stabilizing a preexisting F-actin angular conformation. In addition, ADF induces a large ( approximately 12 degrees ) tilt of actin subunits at high pH where filaments are readily disrupted. A second ADF molecule binds to a site on the opposite side of F-actin from that of the previously described ADF binding site, and this second site is only largely occupied at high pH. All of these states display a high degree of cooperativity that appears to be an integral part of F-actin.     

Cooperative mechanisms in the activation dependence of the rate of force development in rabbit skinned skeletal muscle fibers

Regulation of contraction in skeletal muscle is a highly cooperative process involving Ca(2+) binding to troponin C (TnC) and strong binding of myosin cross-bridges to actin. To further investigate the role(s) of cooperation in activating the kinetics of cross-bridge cycling, we measured the Ca(2+) dependence of the rate constant of force redevelopment (k(tr)) in skinned single fibers in which cross-bridge and Ca(2+) binding were also perturbed. Ca(2+) sensitivity of tension, the steepness of the force-pCa relationship, and Ca(2+) dependence of k(tr) were measured in skinned fibers that were (1) treated with NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1, to promote cooperative strong binding of endogenous cross-bridges to actin; (2) subjected to partial extraction of TnC to disrupt the spread of activation along the thin filament; or (3) both, partial extraction of TnC and treatment with NEM-S1. The steepness of the force-pCa relationship was consistently reduced by treatment with NEM-S1, by partial extraction of TnC, or by a combination of TnC extraction and NEM-S1, indicating a decrease in the apparent cooperativity of activation. Partial extraction of TnC or NEM-S1 treatment accelerated the rate of force redevelopment at each submaximal force, but had no effect on kinetics of force development in maximally activated preparations. At low levels of Ca(2+), 3 microM NEM-S1 increased k(tr) to maximal values, and higher concentrations of NEM-S1 (6 or 10 microM) increased k(tr) to greater than maximal values. NEM-S1 also accelerated k(tr) at intermediate levels of activation, but to values that were submaximal. However, the combination of partial TnC extraction and 6 microM NEM-S1 increased k(tr) to virtually identical supramaximal values at all levels of activation, thus, completely eliminating the activation dependence of k(tr). These results show that k(tr) is not maximal in control fibers, even at saturating [Ca(2+)], and suggest that activation dependence of k(tr) is due to the combined activating effects of Ca(2+) binding to TnC and cross-bridge binding to actin.     

Hidden Markov model analysis of intermediate gating steps associated with the pore gate of shaker potassium channels.

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 micros at -100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a "pore gate" associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.     

Autoreceptors do not regulate routinely neurotransmitter release: focus on adrenergic systems

The theory that neurotransmitter release is regulated locally at the individual terminals of neurons has achieved a rapid and seemingly secure status in our understanding of neuronal function both in the periphery and in the central nervous system. This concept of negative feedback control through the monitoring of the perineuronal concentration of previously released transmitter has been extended to a multiplicity of transmitters and utilized to explain the mechanisms of action of diverse classes of drugs, ranging from antihypertensives to antidepressants. It is my view that negative feedback by terminal and by somadendritic receptors cannot account for the existing body of experimental work. Analyses of the profiles of action of agonists and antagonists, and of the per pulse release of transmitter in the absence of drugs in a variety if peripheral organ systems, as well as in superfused brain slices, demonstrates the need for alternate interpretations of the available data. Evidence is provided that the actions of agonists to inhibit transmitter release and that of antagonists to enhance release occur at different cellular loci and that the purported unitary action of these two classes that is so central to the validity of presynaptic theory is unsupportable.     

Uncoupled Active Transport Mechanisms Accounting for Low Selectivity in Multidrug Carriers: P-Glycoprotein and SMR Antiporters

The extraordinarily low substrate specificity of P-glycoprotein conflicts with the notion that specific substrate interactions are required in the control of the reaction path in an active transport system. The difficulty is shown to be overcome by a half-coupled mechanism in which the ATP reaction is linked to carrier transformations, as in a fully coupled system, but in which the transported substrate plays a passive role. The mechanism, which requires no specific interaction with the substrate, brings about uphill transport. A half-coupled mechanism is directly supported by two observations: (i) almost completely uncoupled ATPase activity in purified P-glycoprotein, and (ii) a pattern of substrate specificity like that of passive systems, where maximum rates for different substrates vary little (unlike active systems, where maximum rates vary greatly). The mechanism accommodates other findings: partial inhibition of ATPase activity by an actively transported substrate; simultaneous binding and translocation of more than one substrate molecule; and stimulation or inhibition of the transport of one substrate molecule by another. A half-coupled system associated with an internal competitive inhibitor should behave as if tightly coupled, in agreement with the effects of the synthetic peptide, polytryptophan. The degree of coupling in the intact system is yet to be determined, however. A half-coupled ATPase mechanism could originally have evolved in a flippase, where immersion of the carrier in its substrate, the membrane lipid, precludes uncoupled ATP hydrolysis. These concepts may have wider application. An uncoupled antiport mechanism, driven by a proton gradient rather than ATP, can explain low selectivity in the SMR multidrug carriers of bacteria, and a half-coupled mechanism for the ion-driven cotransport of water (the substrate in which the carrier site is immersed) can explain a recently proposed uphill flow of water. Received: 23 April 1999/Revised: 29 July 1999     

Improved non-viral transfection of glial and adult neural stem cell lines and of primary astrocytes by combining agents with complementary modes of action

BACKGROUND: Rational design of gene vectors for therapeutic applications requires understanding of transfection mechanisms. In this study, multiple transfection assays revealed complementary mechanisms between two commonly used transfection agents. This finding was then exploited to produce improved transfection outcomes. METHODS AND RESULTS: Rat C6 glial cells, adult rat hippocampal progenitor cells and primary astrocytes were transfected using Lipofectamine (LA) or polyethylenimine (PEI), in vitro. Although LA- and PEI-transfected populations expressed the same total level of transgene product, LA transfected considerably more cells than PEI (approximately 20 vs. 14%). A fluorescently labelled plasmid and time-course analysis, involving both flow cytometry and confocal microscopy, were used to explain this apparent discrepancy. Results showed that LA delivered more plasmid DNA to the cytoplasm and achieved transgene expression in more cells than PEI. In contrast, PEI transfected fewer cells but, on average, produced more transgene product per transfected cell. CONCLUSIONS: A comparative transfection model was developed to explain these different characteristics. According to this model, transfection is a multistage process with different transfection agents exerting their primary effect at different stages in this process. This model forecast that it should be possible to prepare a chimeric complex with a transfection efficiency that exceeded that achievable with Lipofectamine or polyethylenimine alone. This prediction was tested and shown to hold for glioma cells, primary astrocytes, and adult neural stems cells.     

A Non-Native α-Helix Is Formed in the β-Sheet Region of the Molten Globule State of Canine Milk Lysozyme

The native and the molten globule states (N and MG states, respectively) of canine milk lysozyme (CML) were examined by CD spectroscopy and AGADIR algorithm, a helix-coil transition program. It revealed that the helical content of the MG state was higher than that of the N-state, suggesting that non-native alpha-helix is formed in the MG state of CML. Using AGADIR, it indicated the possibility of alpha-helix formation in the third beta-strand region in the MG state. To investigate this possibility, we designed a mutant, Q58P, in which the helical propensity of the MG state was significantly decreased around the third beta-strand region. It appeared that the absolute ellipticity value at 222 nm of the mutant in the MG state was smaller than that of the wild-type protein. It could be assumed that the non-native alpha-helix is formed around the third beta-strand region of wild-type CML in the MG state.