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.     

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.     

An evolutionary perspective on pathogenic mtDNA mutations: haplogroup associations of clinical disorders

More than 75 human diseases have been associated with mitochondrial dysfunction, and many of these are directly caused by overtly pathogenic mutations in the mitochondrial genome (mtDNA). In addition, there have been a number of reports that posit a different, subtler role for mtDNA substitutions in the disease process. As we review here, mtDNA evolution has resulted in the distribution of sequences into continent-specific haplogroups, which are defined by a relatively small number of polymorphisms. Thus, mtDNA sequences can be assigned to European, African, or Asian/Native American haplogroups. There are numerous reports that various diseases are haplogroup-associated, and it has been suggested that some of these haplogroup-associated polymorphisms act as risk factors in these disorders. It has also been suggested that there are haplogroup-associations for aging. As we note here, however, such associations have usually been observed only in single studies and it is difficult to draw broad conclusions on the basis of the available evidence. At a minimum, we suggest that, a haplogroup-group association must be detected in multiple subpopulations or in a large, carefully controlled population survey.     

Comparative Impacts of Heavy Metals on Root Growth as Related to Their Specificity and Selectivity

Two-day-old maize (Zea mays L.) seedlings were incubated on the solutions of Ag, Cd, Pb, Zn, Cu, Tl, Co, and Hg salts (0.001 to 3 g/l). Toxicity of heavy metals was assessed as the inhibition of root growth on the first, second, and third days, the change in the length of the lateral root zone, and the duration of lateral root development from the first division in pericycle to emergence. For all salts under study, the ratio of the lethal concentration to the lowest concentration slowing down root growth was about ten, and growth inhibition was not almost enhanced in the course of three days. With concentrations calculated as g/l, metal toxicity declined in the following order: Cu Tl > Ag > Cd > Hg > Co > Zn > Pb; for molar concentrations, the order was the following: Tl³⁺ > Cu²⁺ > > Ag⁺ > Hg²⁺ Cd²⁺ > Zn²⁺ Pb²⁺ Co²⁺. Duration of lateral root development was least affected by heavy metals. Metal affinity of biological compounds for SH-groups was closely correlated (r = 0.9) with the molar concentration that inhibited primary root growth by 50%. Because of the narrow range of effective concentrations, only slightly increasing inhibition over the exposure time, tolerant root branching, and close relationship between the toxicity and the constant of binding to SH-groups, we conclude that the salts under study exert nonselective inhibition and root growth is slowed down due to the general toxicity of heavy metals rather than selective inhibition of any particular process or processes.     

Inactivation of BK channels by the NH2 terminus of the beta2 auxiliary subunit: an essential role of a terminal peptide segment of three hydrophobic residues

An auxiliary beta2 subunit, when coexpressed with Slo alpha subunits, produces inactivation of the resulting large-conductance, Ca(2+) and voltage-dependent K(+) (BK-type) channels. Inactivation is mediated by the cytosolic NH(2) terminus of the beta2 subunit. To understand the structural requirements for inactivation, we have done a mutational analysis of the role of the NH(2) terminus in the inactivation process. The beta2 NH(2) terminus contains 46 residues thought to be cytosolic to the first transmembrane segment (TM1). Here, we address two issues. First, we define the key segment of residues that mediates inactivation. Second, we examine the role of the linker between the inactivation segment and TM1. The results show that the critical determinant for inactivation is an initial segment of three amino acids (residues 2-4: FIW) after the initiation methionine. Deletions that scan positions from residue 5 through residue 36 alter inactivation, but do not abolish it. In contrast, deletion of FIW or combinations of point mutations within the FIW triplet abolish inactivation. Mutational analysis of the three initial residues argues that inactivation does not result from a well-defined structure formed by this epitope. Inactivation may be better explained by linear entry of the NH(2)-terminal peptide segment into the permeation pathway with residue hydrophobicity and size influencing the onset and recovery from inactivation. Examination of the ability of artificial, polymeric linkers to support inactivation suggests that a variety of amino acid sequences can serve as adequate linkers as long as they contain a minimum of 12 residues between the first transmembrane segment and the FIW triplet. Thus, neither a specific distribution of charge on the linker nor a specific structure in the linker is required to support the inactivation process.     

Optimal conservation effort for a population in a stochastic environment

We study the optimal conservation effort for a population in a fluctuating environment. The survivorship of a population is affected by unpredictable environmental fluctuation (noise) and can be improved by conservation effort accompanied by a cost. The optimal effort level is the one that minimizes the total cost, defined as the weighted sum of the population extinction risk and the economic cost of conservation effort. The optimal effort depends on the variance and the probability distribution of the noise, the relative importance of the population's survival vs. the economic cost, the effectiveness of conservation effort, and the time scope over which we optimize. The analysis of dynamic programming illustrates that the choice of extinction risk function greatly affects the optimal effort level. The conservation effort level that is the best solution of a multiple-year optimization may be higher than that for the corresponding single-year optimization, if the population is relatively safe. However, the conservation level for the multiple-year optimization becomes lower than for the single-year optimization if the population is endangered. In a similar manner, the optimal conservation effort level for the problem with a short time scope is either higher or lower than that for the problem with a long time scope, depending on the extinction risk of the population. Next, for each parameter of the model, we define five different sensitivities of extinction probability or of the total cost. We then study the mean increase in the total cost caused by the uncertainty of parameters. To achieve the best conservation result, we need to invest the limited research effort to the parameter with the largest effect to the optimal effort level, rather than to those with large impacts on the extinction probability or on the total cost. The recommended policy should depend critically on the choice of the criterion to optimize, which shows the importance of theoretical study of the relationship in performing proper decision making in conservation practice.     

Thoughts on thiolate tethering. Tribute and thanks to a teacher

A unique feature of P450 enzymes is in the presence of a thiolate ligand heme but its exact function in catalysis is a matter of debate. For P450 dependent monooxygenases the "active oxygen" complex seems to exist only as a transition state in which the thiolate ligand provides electron density in order to prevent pi-backbonding of the oxygen to the iron (-S-Fe-O(z.rad;)). The corresponding ground state (Compound I) would be a ferryl species (Fe(IV)z.dbnd6;O) with an electron hole either at the porphyrin or at the sulfur. Apart from this role we postulate that a second function is related to the electronic structure of Compound II as an electron acceptor and this property is shared among monooxygenases, thromboxane synthase, prostacyclin synthase, allene oxide synthase, P450(NOR(-)) and chloroperoxidase. As a common step in all P450 enzymes an extremely rapid electron uptake by Compound II allows that the primary substrate radicals are oxidized to cations which immediately combine with a neighbouring nucleophile. Thus "electron transfer" may substitute for "oxygen rebound" as the final step leading to product formation. The same principle also applies methane monooxygenases in which the role of the thiyl sulfur is replaced by a ferryl-oxyl entity.