A re-examination of the role of the nucleus in generating the circadian rhythm in Acetabularia.

The role of the nucleus in the generation of the circadian rhythm in Acetabularia has been nuclear. Early experiments showed that the plant could exhibit a circadian rhythm in the absence of a nucleus. However, other experiments appeared to show that the nucleus could impart phase information to the rhythm, and so therefore must be a part of the system that generates the rhythm. We have conducted experiments similar to these--in particular, one in which the nuclear end of the plant was entrained on a light-dark cycle that was opposite that of the rest of the plant. The phase of the free-running rhythm of this type of plant is not consistent with the conclusion that the nucleus is part of the circadian oscillator. We have also tried entraining opposite ends of plants with no nuclei on opposite light-dark cycles. The ultimate phases of these plants appear to be nearly random. A possible interpretation of these experiments is discussed.     

杂交试验中的最佳样本含量

杂交试验是一项费时费钱的工作,因此在进行试验之前如能进行严密的设计,给出试验所需的样本大小是十分必要的,统计学中常见的估计样本容理的公式不宜应用于杂交试验,本文分两种情况给出了杂交试验中样本容量的估计公式,据此估计出的样本容量安排杂交试验,可在满足试验者要求的条件下,使试验的总成本最低或使试畜的总头数最少。     

Removal of zero-quantum interference in NOESY spectra of proteins by utilizing the natural inhomogeneity of the radiofrequency field

Summary A single scan method for the suppression of signals arising from zero-quantum coherences (ZQC) is analysed with respect to its application to NMR experiments on proteins. The ZQC are dephased during a spinlock period due to the natural RF inhomogeneity of a commercial probe. A quantitative analysis of a ZQC-compensated NOESY experiment is given. Although the build-up curve for the cross peaks in ZQC-compensated NOESY experiments differ from those in uncompensated experiments, interproton distances in medium-sized proteins can be evaluated with high accuracy. The proposed method is compared with other techniques for ZQC suppression.     

Biodiversity–ecosystem function experiments reveal the mechanisms underlying the consequences of biodiversity change in real world ecosystems

In a recent Forum paper, Wardle (Journal of Vegetation Science, 2016) questions the value of biodiversity–ecosystem function (BEF) experiments with respect to their implications for biodiversity changes in real world communities. The main criticism is that the previous focus of BEF experiments on random species assemblages within each level of diversity has ‘limited the understanding of how natural communities respond to biodiversity loss.’ He concludes that a broader spectrum of approaches considering both non‐random gains and losses of diversity is essential to advance this field of research. Wardle's paper is timely because of recent observations of frequent local and regional biodiversity changes across ecosystems. While we appreciate that new and complementary experimental approaches are required for advancing the field, we question criticisms regarding the validity of BEF experiments. Therefore, we respond by briefly reiterating previous arguments emphasizing the reasoning behind random species composition in BEF experiments. We describe how BEF experiments have identified important mechanisms that play a role in real world ecosystems, advancing our understanding of ecosystem responses to species gains and losses. We discuss recent examples where theory derived from BEF experiments enriched our understanding of the consequences of biodiversity changes in real world ecosystems and where comprehensive analyses and integrative modelling approaches confirmed patterns found in BEF experiments. Finally, we provide some promising directions in BEF research.     

Response surfaces for overdispersion in the study of the conditions for fish eggs hatching

Response surface methodology, originally developed for determining optimal conditions in industrial experiments, was early adapted to experiments in marine ecology. However, these involved studying the shape of the complete response surface, not only detecting the optimum, and often had counts or durations as the response variable. Thus, nonlinear, nonnormal response models were required. For counts, binomial and beta-binomial models have been used, the latter because of substantial overdispersion. In closely controlled experiments, overdispersion among units held under the same conditions might indicate that some mishap has occurred in conducting the study. One possible check is to model the dispersion as a second response surface. This procedure is used to show that overdispersion in fish egg hatching experiments has a biological explanation in that it occurs only under suboptimal hatching conditions.     

Selection experiments and the study of phenotypic plasticity1

Abstract Laboratory selection experiments are powerful tools for establishing evolutionary potentials. Such experiments provide two types of information, knowledge about genetic architecture and insight into evolutionary dynamics. They can be roughly classified into two types: (1) artificial selection in which the experimenter selects on a focal trait or trait index, and (2) quasi‐natural selection in which the experimenter establishes a set of environmental conditions and then allows the population to evolve. Both approaches have been used in the study of phenotypic plasticity. Artificial selection experiments have taken various forms including: selection directly on a reaction norm, selection on a trait in multiple environments, and selection on a trait in a single environment. In the latter experiments, evolution of phenotypic plasticity is investigated as a correlated response. Quasi‐natural selection experiments have examined the effects of both spatial and temporal variation. I describe how to carry out such experiments, summarize past efforts, and suggest further avenues of research.     

A geometric approach to determine association and coherence of the activation times of cell-cycling genes under differing experimental conditions

Differing arresting agents and protocols can be used to synchronize cells in cultures to specific phases of the cell when studying cell-cycle gene expressions. Often, data derived from individual experiments are analyzed separately, since no appropriate statistical methodology is available at the moment to analyze the data from all such experiments simultaneously. The focus of this paper is to determine the association and coherence of the relative activation times of cell-cycling genes under different experimental conditions. Using a circular-circular regression model, we define two parameters, a rotation parameter for the angular difference between cells' arresting times (phases) in two cell-cycle experiments, and an association parameter to describe the correspondence between the cycle times of maximal expression (phase angles) for a set of genes studied in two experiments. Further, we propose a procedure to assess coherence across multiple experiments, i.e. to what extent the circular ordering of the phase angles of genes is maintained across multiple experiments. Coherence of genes across experiments suggests that functionally these genes tend to respond in a stereotypically sequenced way under different experimental conditions. Our proposed methodology is illustrated by applying it to a HeLa cell-cycle gene-expression data.     

Charting the history of agricultural experiments

Agricultural experimentation is a world in constant evolution, spanning multiple scientific domains and affecting society at large. Even though the questions underpinning agricultural experiments remain largely the same, the instruments and practices for answering them have changed constantly during the twentieth century with the advent of new disciplines like molecular biology, genomics, statistics, and computing. Charting this evolving reality requires a mapping of the affinities and antinomies at work within the realm of agricultural research, and a consideration of the practices, tools and social and political structures in which agricultural experiments are grounded. Three main questions will be addressed to provide an overview of the complex world of agricultural research investigated by the special issue: What is an agricultural experiment? Who is an experimenter in agriculture? Where do agricultural experiments take place? It will become apparent that agricultural experiments have a wide relevance for human development as they touch upon concerns related to human health and nutrition, contribute to policy discussions, and can affect the social and political structures in which farming is embedded.

     

Exploration of plant growth and development using the European Modular Cultivation System facility on the International Space Station

Space experiments provide a unique opportunity to advance our knowledge of how plants respond to the space environment, and specifically to the absence of gravity. The European Modular Cultivation System (EMCS) has been designed as a dedicated facility to improve and standardise plant growth in the International Space Station (ISS). The EMCS is equipped with two centrifuges to perform experiments in microgravity and with variable gravity levels up to 2.0 g. Seven experiments have been performed since the EMCS was operational on the ISS. The objectives of these experiments aimed to elucidate phototropic responses (experiments TROPI‐1 and ‐2), root gravitropic sensing (GRAVI‐1), circumnutation (MULTIGEN‐1), cell wall dynamics and gravity resistance (Cell wall/Resist wall), proteomic identification of signalling players (GENARA‐A) and mechanism of InsP₃ signalling (Plant signalling). The role of light in cell proliferation and plant development in the absence of gravity is being analysed in an on‐going experiment (Seedling growth). Based on the lessons learned from the acquired experience, three preselected ISS experiments have been merged and implemented as a single project (Plant development) to study early phases of seedling development. A Topical Team initiated by European Space Agency (ESA), involving experienced scientists on Arabidopsis space research experiments, aims at establishing a coordinated, long‐term scientific strategy to understand the role of gravity in Arabidopsis growth and development using already existing or planned new hardware.     

3H]phenamil, a radiolabelled diuretic for the analysis of the amiloride-sensitive Na+ channels in kidney membranes

The interaction of amiloride and amiloride derivatives with the Na+ channels of pig kidney membranes was studied from 22Na+ uptake experiments. The order of potency of the different molecules tested is: phenamil greater than benzamil greater than amiloride, ethylisopropylamiloride. [3H]labelled phenamil was prepared and used to titrate Na+ channels in pig kidney membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]phenamil and unlabelled phenamil indicate that phenamil recognizes a single family of binding sites with a Kd value of 20 nM and a maximum binding capacity of 11.5 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]phenamil competition experiments is identical to that found for the inhibition of 22Na+ uptake by apical Na+ channels.     

Fluorescent protein FRET: the good, the bad and the ugly

Dynamic protein interactions play a significant part in many cellular processes. A technique that shows considerable promise in elucidating such interactions is F?rster resonance energy transfer (FRET). When combined with multiple, colored fluorescent proteins, FRET permits high spatial resolution assays of protein-protein interactions in living cells. Because FRET signals are usually small, however, their measurement requires careful interpretation and several control experiments. Nevertheless, the use of FRET in cell biological experiments has exploded over the past few years. Here we describe the physical basis of FRET and the fluorescent proteins appropriate for these experiments. We also review the approaches that can be used to measure FRET, with particular emphasis on the potential artifacts associated with each approach.     

Simple experiments to understand the ionic origins and characteristics of the ventricular cardiac action potential

Electrophysiological experiments are helpful for students to understand the role of electrical activity in heart function. Papillary muscle, which belongs to the ventricle, offers the advantage of being easily studied using glass microelectrodes. In addition, there is commercially available software that simulates ventricular electrical activity and can help overcome some difficulties, such as voltage clamp experiments, which need expensive apparatus when used for studies on living preparations. Here, we present a class practical session that is taken by undergraduate students at our University. In the first part of this class, students record action potentials from papillary muscles with the use of glass microelectrodes, and they change extracellular conditions to study the ionic basis of the action potential. In the second part of the class, students simulate action potentials using the Oxsoft Heart model (v. 4.0) and model their previous experiments on papillary muscle to quantify the effects. In particular, the model is very helpful in promoting understanding of the effect that extracellular potassium has on cardiac action potential by simulating voltage clamp experiments. This twin approach of papillary muscle experiments and computer modeling leads to a good understanding of the functioning of the action potential and can help introduce discussion of some abnormal cardiac functioning.     

Separation of the rotational contribution in fluorescence correlation experiments

The theory of fluorescence correlation spectroscopy is reexamined with the aim of separating the contribution of rotational diffusion. Under constant excitation, fluorescence correlation experiments are characterized by three polarizations: one of the incident beam and two of the two photon detectors. A set of experiments of different polarizations is proposed for study. From the results of the experiments the isotropic factor of the fluorescence intensity correlation functions can be determined, which is independent of the rotational motion of the sample molecule. This function can be used to represent each fluorescence intensity correlation function as the product of the isotropic and the rotational factors. The theory is illustrated by an experiment in which rotational diffusion of porcine pancreatic lipase labeled with Texas Red was observed Texas Red is a label that allows precise fluorescence correlation experiments even in the nanosecond time range.     

Interaction of telomestatin with the telomeric single-strand overhang

The extremities of chromosomes end in a G-rich single-stranded overhang that has been implicated in the onset of the replicative senescence. The repeated sequence forming a G-overhang is able to adopt a peculiar four-stranded DNA structure in vitro called a G-quadruplex, which is a poor substrate for telomerase. Small molecule ligands that selectively stabilize the telomeric G-quadruplex induce telomere shortening and a delayed growth arrest. Here we show that the G-quadruplex ligand telomestatin has a dramatic effect on the conformation of intracellular G-overhangs. Competition experiments indicate that telomestatin strongly binds in vitro and in vivo to the telomeric overhang and impairs its single-stranded conformation. Long-term treatment of cells with telomestatin greatly reduces the G-overhang size, as evidenced by specific hybridization or telomeric oligonucleotide ligation assay experiments, with a concomitant delayed loss of cell viability. In vivo protection experiments using dimethyl sulfate also indicate that telomestatin treatment alters the dimethyl sulfate effect on G-overhangs, a result compatible with the formation of a local quadruplex structure at telomeric overhang. Altogether these experiments strongly support the hypothesis that the telomeric G-overhang is an intracellular target for the action of telomestatin.     

Statistical approach to combining the results of similar experiments, with application to the hematologic effects of extremely-low-frequency electric field exposures

A large proportion of scientific effort in investigating the possible biological effects of exposure to extremely-low-frequency (ELF) fields consists of laboratory studies on experimental animals. Most experiments in which hematologic properties are measured show no statistically significant effect due to exposure. However, some studies show significant effects which, in general, are not clearly reproducible. A difficult question must then be addressed: Are these relatively few indications of ELF effects statistical artifacts due to the increased risk of a type I error in multiple studies, or is there a real biological effect that is undetected in most studies due to the relatively small sample sizes commonly used? A statistical approach for examining the accumulated results of multiple experiments which results in a single test for treatment effect is presented. The technique requires very mild assumptions, and is valid for experiments that vary widely in specific characteristics such as exposure level, duration, and laboratory. The method is applied to the results of a collection of hematologic and serum chemistry experiments, and the combined results indicate the existence of experimental effects on some end points.     

On the Physical Basis of the Amino Acid Polar Requirement

Understanding how codons became associated with their specific amino acids is fundamental to deriving a theory for the origin of the genetic code. Carl Woese and coworkers designed a series of experiments to test associations between amino acids and nucleobases that may have played a role in establishing the genetic code. Through these experiments it was found that a property of amino acids called the polar requirement (PR) is correlated with the organization of the codon table. No other property of amino acids has been found that correlates with the codon table as well as PR, indicating that PR is uniquely related to the modern genetic code. Using molecular dynamics simulations of amino acids in solutions of water and dimethylpyridine used to experimentally measure PR, we show that variations in the partitioning between the two phases as described by radial distribution functions correlate well with the measured PRs. Partition coefficients based on probability densities of the amino acids in each phase have the linear behavior with base concentration as suggested by PR experiments.     

Analysis of the oligomeric structure of the motor protein prestin

Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed approximately 11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an approximately 11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestin-prey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the approximately 11-nM particles in the outer hair cell's basolateral membrane.     

On the analysis of the cat's pattern recognition system

The objective of the paper is to determine in abstract terms the algorithms used by the cat detecting simple patterns and to quantify the contributions of the visual areas 17, 18, 19 for this task. The data incorporated in the algorithm are collected from behavioral experiments where the animals had to distinguish between two patterns. The patterns were superimposed with gaussian noise and the detection probability was measured. The resulting model describes pattern recognition in two steps: first extraction of features and second classification. The test of the validity of the model system was to predict the outcome of similar experiments but with different patterns. With the help of the model it is possible to describe the effect of a lesion in parametric form. This in turn gives some hints about the functions of the visual areas 17, 18, 19 for the specific fask, tested in the experiments.     

Development and verification of hardware for life science experiments in the Japanese Experiment Module "Kibo" on the International Space Station

Japan Aerospace Exploration Agency (JAXA) has developed a cell biology experiment facility (CBEF) and a clean bench (CB) as a common hardware in which life science experiments in the Japanese Experiment Module (JEM known as "Kibo") of the International Space Station (ISS) can be performed. The CBEF, a CO2 incubator with a turntable that provides variable gravity levels, is the basic hardware required to carry out the biological experiments using microorganisms, cells, tissues, small animals, plants, etc. The CB provides a closed aseptic operation area for life science and biotechnology experiments in Kibo. A phase contrast and fluorescence microscope is installed inside CB. The biological experiment units (BEU) are designed to run individual experiments using the CBEF and the CB. A plant experiment unit (PEU) and two cell experiment units (CEU type1 and type2) for the BEU have been developed.     

提高植物学实验教学质量的一些体会

实验是植物学教学的重要环节.针对植物学传统实验教学中的实际情况,进行了实验方法力求多样化、变部分过程性实验为过程性实验、增加综合性实验和研究性实验等尝试,进而充分发挥教师和学生在实验教学中的作用,培养学生的创造能力.