土壤优先水流及溶质优先迁移的研究

通过土壤原状土柱和填充土柱实验,研究了土壤优先水流的特征和非吸附性离子Br^-和NO3^-在土壤中的优先迁移。结果表明,土壤优先水流存在优先穿透、穿透曲线不对称性、偶向入渗和拖尾等特征,原状土柱中Br^-标记的优先水流在实验开始后24h出现穿透,穿透时的孔隙体积仅0.04,在原状土柱中以Br^-标记的土壤优先水流占土壤出流总量的26％,所引起的Br^-累积淋出量占总淋出量的86.7％,说明土壤优先水流虽只占出流总量较小的比例,却可造成较大比例的溶质迁移,优先水流可使NO3^－快速向下迁移,在仅1倍孔隙体积时,其出流中可收获投加量的11％,优先水流可使Br^-和NO3^-向土壤较深层次迁移的同时,发生径向扩散。     

Endogenous respiration and regulation in a primitive marine fungus

The relationship between the respiration and the presence and utilization of endogenous and exogenous substrates was studied in the non-filamentous obligately marine fungus Thraustochytrium aureum. Using isotopic and manometric methods, it was shown that almost all exogenous glucose is assimilated, whilst almost all the oxygen consumption in the presence of exogenous glucose was due to oxidation of endogenous reserves. In contrast, exogenous glutamate, which cannot serve as the sole carbon source for growth, inhibits respiration of endogenous materials, and is itself rapidly oxidized. The uncoupler 2,4-dinitrophenol stimulates the oxidation of endogenous reserves without affecting the uptake and use of exogenous glucose. These data strongly support the idea of physiologic compartmentation in this organism.     

Regulation of acid hydrolysis of sucrose in acid lime juice sac cells

The sucrose content of acid lime [ Citrus aurantifolia (Christm.) Swing.] juice tissue was measured at time 0 and at various times following incubation at 15.5, 26.6 and 37.7°C. The decline in sucrose content in fruit stored at 15.5°C paralleled the expected values for a sucrose solution at pH 2.1. At higher temperatures, the in vivo sucrose content decreased at significantly lower rates than the expected values. In fruit stored at 26.6 and 37.7°C, the vacuolar pH increased 0.11 and 0.23 units, respectively. When sucrose hydrolysis was recalculated at the increased vacuolar pH of juice cells stored at 26.6 and 37.7°C, the calculated values were similar to the measured values obtained in vivo. It is concluded that within the limits of the experimental conditions, the rates of sucrose acid hydrolysis are regulated by changes in the vacuolar H⁺ concentration.     

Dual-porosity model of solute diffusion in biological tissue modified by electroporation

In many electroporation applications mass transport in biological tissue is of primary concern. This paper presents a theoretical advancement in the field and gives some examples of model use in electroporation applications. The study focuses on post-treatment solute diffusion.     

Lack of the Golgi phosphate transporter PHT4;6 causes strong developmental defects,constitutively activated disease resistance mechanisms and altered intracellular phosphate compartmentation in Arabidopsis

The Golgi‐located phosphate exporter PHT4;6 has been described as involved in salt tolerance but further analysis on the physiological impact of PHT4;6 remained elusive. Here we show that PHT4;6–GFP is targeted to the trans‐Golgi compartment and that loss of function of this carrier protein has a dramatic impact on plant growth and development. Knockout mutants of pht4;6 exhibit a dwarf phenotype that is complemented by the homologous gene from rice (Oryza sativa). Interestingly, pht4;6 mutants show altered characteristics of several Golgi‐related functions, such as an altered abundance of certain N‐glycosylated proteins, altered composition of cell‐wall hemicelluose, and higher sensitivity to the Golgi α‐mannosidase and the retrograde transport inhibitors kifunensine and brefeldin A, respectively. Moreover, pht4;6 mutants exhibit a ‘mimic disease’ phenotype accompanied by constitutively activated pathogen defense mechanisms and increased resistance against the virulent Pseudomonas syringae strain DC3000. Surprisingly, pht4;6 mutants also exhibit phosphate starvation symptoms, as revealed at the morphological and molecular level, although total Pi levels in wild‐type and pht4;6 plants are similar. This suggested that subcellular Pi compartmentation was impaired. By use of nuclear magnetic resonance (NMR), increased Pi concentration was detected in acidic compartments of pht4;6 mutants. We propose that impaired Pi efflux from the trans‐Golgi lumen results in accumulation of inorganic phosphate in other internal compartments, leading to low cytoplasmic phosphate levels with detrimental effects on plant performance.     

Determination of the cytosolic free NAD/NADH ratio in Saccharomyces cerevisiae under steady-state and highly dynamic conditions

The coenzyme NAD plays a major role in metabolism as a key redox carrier and signaling molecule but current measurement techniques cannot distinguish between different compartment pools, between free and protein-bound forms and/or between NAD(H) and NADP(H). Local free NAD/NADH ratios can be determined from product/substrate ratios of suitable near-equilibrium redox reactions but the application of this principle is often precluded by uncertainties regarding enzyme activity, localization and coenzyme specificity of dehydrogenases. In Saccharomyces cerevisiae, we circumvented these issues by expressing a bacterial mannitol-1-phosphate 5-dehydrogenase and determining the cytosolic free NAD/NADH ratio from the measured [fructose-6-phosphate]/[mannitol-1-phosphate] ratio. Under aerobic glucose-limited conditions we estimated a cytosolic free NAD/NADH ratio between 101(+/-14) and 320(+/-45), assuming the cytosolic pH is between 7.0 and 6.5, respectively. These values are more than 10-fold higher than the measured whole-cell total NAD/NADH ratio of 7.5(+/-2.5). Using a thermodynamic analysis of central glycolysis we demonstrate that the former are thermodynamically feasible, while the latter is not. Furthermore, we applied this novel system to study the short-term metabolic responses to perturbations. We found that the cytosolic free NAD-NADH couple became more reduced rapidly (timescale of seconds) upon a pulse of glucose (electron-donor) and that this could be reversed by the addition of acetaldehyde (electron-acceptor). In addition, these dynamics occurred without significant changes in whole-cell total NAD and NADH. This approach provides a new experimental tool for quantitative physiology and opens new possibilities in the study of energy and redox metabolism in S. cerevisiae. The same strategy should also be applicable to other microorganisms.     

Die Entwicklung der Zellforschung

Obwohl erst im 17. Jahrhundert begonnen, hat die Zellforschung doch bald zur Formulierung allgemein-biologischer Theorien geführt und trägt auch heute ständig weiter zu fundamentalen Theorienbildungen bei. Die Entwicklung der Zellforschung verdient daher als Paradigma für die Gewinnung naturwissenschaftlicher Einsichten besonderes Interesse: An ihr können Grundbedingungen wissenschaftlichen Erkenntnisfortschrittes gut verdeutlicht werden als Wechselspiel von Methodenerweiterung und Konzeptualisierung des Neuentdeckten. Erst eineinhalb Jahrhunderte nach der Erstbeobachtung von Zellen gelang die Formulierung der “Zellentheorie”. Auf der durch sie geschaffenen, konzeptionellen Grundlage war es vielen biologischen und medizinischen Disziplinen möglich, von einem beobachtend-beschreibenden (“induktiven”) zu einem experimentierenderklärenden (“deduktiven”) Status vorzustoßen. Ein außergewöhnlicher Erfolg war dabei der Cytogenetik beschieden. Dagegen war der Versuch, in den subzellulären und submikroskopischen Bereich vorzudringen, nur teilweise erfolgreich (Polarisationsmikroskopie, Nägelis Micellartheorie). Ein breiter Durchbruch gelang in diesem Sektor erst um 1950 durch eine beinahe einmalige zeitliche Kumulierung methodischer Fortschritte, wobei vor allem Zellfraktionierung und Elektronenmikroskopie eine entscheidende Rolle spielten. Zusammen mit der vorausgegangenen Entwicklung “flankierender” Wissenschaften (insbesondere der Makromolekularen Chemie) wurde so die Etablierung der modernen Zellbiologie möglich. Einige ihrer bedeutendsten Aussagen werden diskutiert (Zellkompartimentierung und genetische Kontinuität ohne Nucleinsäuren; Zweiteilung der Organismenwelt nach der Zellorganisation; Evolution zellulärer Organismen und Symbiontentheorie). Abschließend wird versucht, Bedingungen und Erfordernisse von wissenschaftlichem Fortschritt allgemein zu formulieren und daran die heutige Wissenschaftswirklichkeit kritisch zu messen.     

Regulation of cytokinin content in plant cells

Cytokinin levels in plant cells are dependent on cytokinin biosynthesis and/or uptake from extracellular sources, metabolic interconversions, inactivation and degradation. Cytokinin conversion to compounds differing in polarity seems to be decisive for their entrapment within the cell and intracellular compartmentation, which affects their metabolic stability. Increase in cytokinin levels, resulting either from their uptake or intracellular biosynthesis, may promote further autoinductive accumulation of cytokinins which may function in the induction of cytokinin-initiated physiological processes. Accumulated cytokinins are capable of inducing cytokinin oxidase which consequently decreases cytokinin levels. This seems to be the mechanism of re-establishment and maintenance of cytokinin homeostasis required for further development of physiological events induced by transient cytokinin accumulation. Auxin may influence cytokinin levels by down regulation of cytokinin biosynthesis and/or by promotion of cytokinin degradation. A model of the regulation of cytokinin levels in plant cells based on these phenomena is presented and its physiological role(s) is discussed.     

Hexokinase Binding to Mitochondria: A Basis for Proliferative Energy Metabolism

Current thought is that proliferating cells undergo a shift from oxidative to glycolytic metabolism, where the energy requirements of the rapidly dividing cell are provided by ATP from glycolysis. Drawing on the hexokinase–mitochondrial acceptor theory of insulin action, this article presents evidence suggesting that the increased binding of hexokinase to porin on mitochondria of cancer cells not only accelerates glycolysis by providing hexokinase with better access to ATP, but also stimulates the TCA cycle by providing the mitochondrion with ADP that acts as an acceptor for phosphoryl groups. Furthermore, this acceleration of the TCA cycle stimulates protein synthesis via two mechanisms: first, by increasing ATP production, and second, by provision of certain amino acids required for protein synthesis, since the amino acids glutamate, alanine, and aspartate are either reduction products or partially oxidized products of the intermediates of glycolysis and the TCA cycle. The utilization of oxygen in the course of the TCA cycle turnover is relatively diminished even though TCA cycle intermediates are being consumed. With partial oxidation of TCA cycle intermediates into amino acids, there is necessarily a reduction in formation of CO₂ from pyruvate, seen as a relative diminution in utilization of oxygen in relation to carbon utilization. This has been assumed to be an inhibition of oxygen uptake and therefore a diminution of TCA cycle activity. Therefore a switch from oxidative metabolism to glycolytic metabolism has been assumed (the Crabtree effect). By stimulating both ATP production and protein synthesis for the rapidly dividing cell, the binding of hexokinase to mitochondrial porin lies at the core of proliferative energy metabolism. This article further reviews literature on the binding of the isozymes of hexokinase to porin, and on the evolution of insulin, proposing that intracellular insulin-like proteins directly bind hexokinase to mitochondrial porin.