Modified cobalt staining and silver intensification techniques for use with whole-mount gastropod ganglion preparations

While silver intensification of cobalt-filled cells is a common procedure for use with many arthropod preparations, it has not been routinely applied to gastropods. Several modifications in cobalt-staining techniques currently used with gastropods along with adaptations of silver intensification techniques used in insects are described. Cobalt was introduced into cells through axonal filling of cut nerve trunks or by either pressure injection or iontophoresis from intracellular, micropipette electrodes that had previously had their tips etched in dilute hydrofluoric acid. Etching produced consistent, sharp tips with large lumina. Further procedural modifications allowed complete, even intensification of neurons in large gastropod ganglia. These techniques have proved to be reliable and apparently broadly applicable, having been successfully used on three diverse gastropod species.     

Electron microscopical demonstration of sulphated mucopolysaccharides in mouse tracheal cartilage with a diaminobenzidine-osmium tetroxide technique

Synopsis A diaminobenzidine-osmium tetroxide method for the demonstration of sulphated mucopolysaccharides has been tested at the electron microscopical level. The reaction (which involves treatment with diaminobenzidine in an acidic solution followed by oxidation with osmium tetroxide) has been carried out directly on ultra-thin sections of mouse tracheal cartilage embedded in water-soluble glycolmethacrylate. Sulphated mucopolysaccharides in the cartilage matrix were localized as a heavy, electron-dense precipitate. The method can be applied directly to sections, overcoming in this way the difficulties of penetration, and it seems to possess a higher specificity for sulphated mucopolysaccharides than that displayed by other techniques proposed previously for the ultrastructural demonstration of acid mucopolysaccharides.     

Directly probing rapid membrane protein dynamics with an atomic force microscope: a study of light-induced conformational alterations in bacteriorhodopsin.

This paper demonstrates that an atomic force microscope can be used to directly monitor rapid membrane protein dynamics. For this demonstration the membrane-bound proton pump, bacteriorhodopsin, has been investigated. It has been unequivocally shown that the light-induced dynamic alterations that have been observed do not arise from external artifacts such as heating of the sample by the incident light, but that these changes can be directly linked to the light-induced protein conformational alterations in this membrane. In essence, it has been shown that the light energy absorbed by bacteriorhodopsin is converted not only to chemical energy but also to mechanical energy. In summary a new ultrasensitive tool is described for monitoring the molecular dynamics of materials with wide applicability to fundamental and applied science.     

黑腹果蝇细胞谱系分析方法进展

对动物体内单个细胞的谱系进行分析有助于追踪其在发育过程中的作用,但是体内各种组织都是由很多形态、结构、功能各不相同的细胞构成的复杂系统,这种复杂性严重阻碍了对单个细胞的研究。嵌合克隆技术(Mosaic technique)和标记技术(Labeling technique)的出现为这一研究提供了强有力的手段。文章介绍了近几年来黑腹果蝇(Drosophila melanogaster)研究中常用的7种嵌合克隆标记方法,包括FRT介导的有丝分裂重组(FRT-mediated mitotic recombination)、MARCM(Mosaic analysis with a repressible cell marker)、TSG(Twin spotgenerator)、Twin-spot MARCM、Q-MARCM(Q system-based MARCM)、Coupled MARCM和G-TRACE(Gal4technique for real-time and clonal expression)技术,详述了这些技术的原理及应用,并对不同技术进行了对比。运用这些技术研究者可以从单细胞水平进行遗传学标记和操作,特别是在神经系统等复杂系统中追踪单个细胞的发育过程。果蝇中的这些技术也将为其他模式生物追踪细胞谱系提供参考。     

“Bottom-up” approach for implementing nano/microstructure using biological and chemical interactions

The “Bottom-up” approach for implementing nano/microstructure using biological self-assembled systems has been investigated with tremendous interest by many researchers in the field of medical diagnostics, material synthesis, and nano/microelectronics. As a result, the techniques for achieving these systems have been extensively explored in recent years. The developed or developing techniques are based on many interdisciplinary areas such as biology, chemistry, physics, electrical engineering, mechanical engineering, and so on. In this paper, we review the fundamentals behind the self-assembly concepts and describe the state of art in the biological and chemical self-assembled systems for the implementation of nano/microstructures. These structures described in the paper can be applied to the implementation of hybrid biosensors, biochip, novel bio-mimetic materials, and nano/microelectronic devices.     

An Example of Circular Statistics in Chronobiological Studies: Analysis of Polymorphism in the Emergence Rhythms of Schistosoma Mansoni Cercariae

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433–451, 2000)     

Circadian clocks: what makes them tick?

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals. (Chronobiology International, 17(4), 433-451, 2000)     

Dissecting the Rhizosphere complexity: The truffle-ground study case

The word Rhizosphere describes the part of the soil which is immediately adjacent to and affected by plant roots. This is a very dynamic environment where plants, soil and microorganisms interact. The plant releases, in addition to biologically active substances, nutritive substances (exudates), which create a privileged habitat for many microbial populations. The same microbes that live in the rhizosphere may be useful for the plant. Interest in this fascinating environment has increased over the years. However, our knowledge of the biology and diversity of microbial populations in the rhizosphere is still limited, because it has always been linked to traditional culture-based techniques. These methods, which only allow the study of cultured microorganisms, do not allow the majority of the organisms existing in nature to be characterized. Over the last few years, this limitation has been overcome through the introduction of methodologies that are independent of culture techniques. This different approach, which has revolutionized scientific research, is known as metagenomics. In this review, the rhizosphere environment is considered with particular attention to the fungal and symbiotic organisms, which populate it. The new environmental genomic techniques and how these have been applied to the study of the various environments and the rhizosphere are described. Finally, a specific rhizosphere, a truffle-ground, is described as our study case.      

Low-cost image processing system for analyzing molecular RNA fingerprints.

A complete image digitizing and processing system is described for capturing, enhancing and analyzing molecular fingerprints. The low-cost, high-resolution system features a Motorola 68000 processor, multi-tasking, a separate video coprocessor, and color or gray scale processing. Thousands of manipulations are possible using functions which include histographic equalization, edge detection, filtering, overlays, false coloring, zoom, pan and print. All operations are initiated and controlled with a mouse. Techniques for enhancing, scaling and comparing molecular fingerprints are described. The techniques all involve using a graphical interface to select and manipulate the various processes. The system has been used successfully for about 1.5 years, and it has been ideal for our application which requires human judgment at many steps between processing and which probably would not lend itself to a completely automated analysis. Similar techniques could probably be used with this system on many other applications.     

Diaspore and gap availability are limiting species richness in wet meadows

Species pool theory claims that diaspore and microsite availability limit species richness in plant communities. Wet meadows (Calthion) and litter meadows (Molinion, Caricion davallianae) belonging to the most species-rich meadows in the foothills of the Alps have suffered from a strong decrease since the 1970s. Restoration efforts including nutrient impoverishment and rewetting management after drainage and fertilization did not result in the re-establishment of the former species richness although the abiotic filter would have been appropriate for the re-colonization of many locally extinct species. In our experiment at four sites in the largest fen of Southwest-Germany we tested if the restoration success was seed- and gap-limited. We applied sowing and hay spreading (hay seed) as treatments to make seeds available and harrowing to increase gap availability. Sowing seeds or hay seed of species of the former meadow types increased species richness immediately. The proportion of re-established species was higher when additional harrowing was applied. Species richness could be increased not only in vascular plants but also in bryophytes when hay spreading was applied. The strongest re-development towards the target communities (defined through the abiotic filter and the species richness before drainage and fertilization) took place on those sites where hay spreading and harrowing were applied. Sowing seeds and hay seed were traditional techniques to establish e.g. litter meadows, both techniques have been applied for centuries. Even harrowing was described as early as the 19th century to increase the chance of establishing certain species. Therefore, the “application of the knowledge coming from the species pool theory” (although not named during this time) has been common practice since at least the 19th century.     

Two-dimensional spectroscopy of electrophoretic gels

Optical techniques are described which permit one to analyze two-dimensional electrophoretic gels in a fashion which is analogous to the one-dimensional spectroscopy of solutions. In the methods described, an electrophoretic gel is irradiated with monochromatic light and isozyme patterns are detected by the absorption of light or the fluorescent emission of light. The system described can both generate and detect monochromatic light in a range from 200 to 1100 nm. Without the use of histochemical stains, several isozymes have been visualized by purely optical means. Five methods for the visualization of lactate dehydrogenase and five methods for the demonstration of trypsin isozymes are described. In addition, general methods have been formulated for hydrolases and oxidases. Gel spectroscopy should permit the investigation of a wide range of new isozymes.This work was supported in part by NIH Grants CA 19017 and GM 21433.     

Discussion of errors and measuring strategies in morphometry using analysis of variance

Statistical techniques known as the analysis of variance make it possible for the morphologist to plan work in such a way as to get quantitative data with the greatest possible economy of effort. This paper explains how to decide how many measurements to make per micrograph, how many micrographs per tissue block or organ, and how many organs or individuals are necessary for getting an exactness of sufficient quality of the results. The examples furnished have been taken from measuring volume densities of mitochondria in heart muscle cells and from cell counting in lymph nodes. Finally we show, how to determine sample sizes, if we are interested in demonstration of significant differences between mean values.     

An X-ray microanalytical study of the ferricyanide reaction for the electron cytochemical demonstration of succinate dehydrogenase activity in isolated mitochondria

Synopsis The cytochemical technique for the demonstration of succinate dehydrogenase activity by ferricyanide reduction and simultaneous coupling with Cu²⁺ has been investigated by the combined techniques of transmission electron microscopy and X-ray microanalysis. The localization of the final reaction product of succinate dehydrogenase activity within isolated mouse liver mitochondria is described, and variations in the composition of the final reaction product are shown to occur, both at various sites within the mitochondrion, as well as after different reaction times.     

一种适用于线粒体基因表达分析的cDNA-RAPD方法

由于植物线粒体DNA分子结构复杂,并在与细胞核共进化的过程中形成了自己独特的表达系统,迄今仍没有一种较好的能够对植物线粒体基因表达进行分析的方法。本文依据线粒体RNA的自身特点,对已用于分析真核mRNA的差展方法进行了改进。采用随机六聚体引物取代oligo(dT)n,从而将线粒体RNA及其他各类无poly(A)尾的mRNA纳入到可直接研究的范围,发展了一种适用于线粒体基因表达分析的方法。 Abstract:Several techniques are available in detecting variations in gene expression between different samples,such as SSH,RACE etc.However,they can not be applied to analyze mitochondrial gene expression due to the specific characteristics of mitochondrial RNA.So some modifications were made to the conventional techniques.Here we reported a demonstration of this modified technique,taking rice mitochondria as materials.In this technique,using random hexamers to prime the RT,the resultant cDNA likely included coding regions because it was not locked to the poly(A) tail of the messenger RNA.     

Protein glycosylation analysis with capillary-based electromigrative separation techniques

An impressive complexity is associated with glycoproteins due to the microheterogeneity of glycosylation as posttranslational modification giving rise to a vast number of isoforms. The full characterization of glycoproteins is difficult to achieve, and a number of analytical methods have to be combined for a detailed understanding of glycosylation. In this review, we focus on capillary electromigrative separation techniques in the formats capillary electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. These separation techniques can be applied to all levels of glycosylation analysis including intact glycoproteins, glycopeptides, and released glycans. We here discuss the separation characteristics for each method and the information that they can provide for each level. Detection issues, especially laser-induced fluorescence detection and mass spectrometry are taken into account. In addition, tables provide an overview on the achievements made from the very beginning of glycosylation research by electromigrative separation techniques. From the literature presented here it is clear, that glycosylation analysis by electromigrative separation techniques is on the edge of transition of basic research and method development towards applications. First proof-of-principle studies for in-depth glycoprotein characterization and clinical diagnosis are described. However, this overview also shows that many basic aspects of separation have not yet been fully understood and more research is necessary to be able to fully use the capabilities of electromigrative separation techniques.     

Commercial high-throughput sequencing and its applications in DNA analysis

DNA sequences can be used for the analysis of genetic variation and gene function. The high-throughput sequencing techniques that have been developed over the past three years can read as many as one billion bases per run, and are far less expensive than the traditional Sanger sequencing method. Therefore, the high-throughput sequencing has been applied extensively to genomic analyses, such as screening for mutations, construction of genomic methylation maps, and the study of DNA-protein interactions. Although they have only been available for a short period, high-throughput sequencing techniques are profoundly affecting many of the life sciences, and are opening out new potential avenues of research. With the highly-developed commercial high-throughput sequencing platforms, each laboratory has the opportunity to explore this research field. Therefore, in this paper, we have focused on commercially-popular high-throughput sequencing techniques and the ways in which they have been applied over the past three years.     

A Reliable Method for Demonstrating Gram-Positive and Gram-Negative Bacteria

Combined Gram techniques have been reviewed in the interest of improving technical safety and reliability in the demonstration of bacteria, particularly the Gram-negative type. The many modifications of the technique present various difficulties (Brown and Brenn 193 1, Humberstone 1963, Taylor 1966, Luna 1968, Brown and Hopps 1973, Engbaek et al. 1979, Bancroft and Stevens 1982, Churukian and Schenk 1982).     

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.     

培养条件下发菜的形态建成

固体培养基培养的发菜(Nostoc flagelliforme)在黑暗与低光强(2·s)条件下细胞发育受到抑制,在光强10、20、30、60 μmol/m2·s 条件下细胞生长良好, 但在60 μmol/m2·s 条件下藻丝体易变黄; 液体充气培养的发菜在光强20、60、180 μmol/m2·s 条件下生长速率、类胡萝卜素与多糖含量均随光强升高而增加。发菜在低营养水平时形成的异形胞较多, 异形胞的发生位置也多样, 有端生、间生和连生。当琼脂浓度为0.5%—4%时发菜具有相同的形态发育特征, 从藻殖段发育至丝状聚集体状态, 再从聚集体中释放藻丝; 当琼脂浓度为6%—8%时发菜发育至丝状聚集体状态, 藻丝包裹在厚胶鞘中, 观察不到藻丝和藻殖段的释放。以上结果表明光照和培养基的含水量对发菜细胞发育具有重要影响。       

萜类生物合成的基因操作

萜类是一组结构迥异的化合物家族,其中很多具有较大的应用价值,如青蒿素和紫杉醇等,它们在多种微生物和植物中合成,但其天然产量低。萜类代谢工程通过DNA重组技术改造萜类合成细胞中的代谢途径,以提高萜类最终产量或在不含萜类的生物中合成萜类,为促进有用萜类合成提供了新的机会。以萜类化合物生物合成途径的基因转移与表达为切入点,综述了目前在微生物及植物中应用代谢工程提高萜类产量的研究进展。