Analysis of enzyme activity regulation by non-denaturing electrophoresis and application of this regulation for enzyme reactor production

Non-denaturing electrophoresis can be used to screen enzymes that self-regulate their activities by using a combination of enzymes and their inhibitors. Furthermore, this technique can be applied to develop enzyme reactors that self-regulate their activities. After separation of proteins from mouse liver cytosol by non-denaturing isoelectric focusing, lactate dehydrogense (LDH) and esterase activities were qualitatively and quantitatively examined using a combination of two-dimensional electrophoresis (2-DE) and non-denaturing stacking gel electrophoresis. Activities of mouse liver-derived LDH and carboxylesterase were reversibly inhibited by oxamate and 6,9-diamino-2-ethoxyacridine (acrinol), respectively, in the stacking gels and recovered when the enzymes migrated towards the separation gels. After separation and immobilization of the enzymes, their activities were inhibited by inhibitors and recovered after inhibitor removal. These results indicate that non-denaturing electrophoresis can be applied to select enzymes that self-regulate their activities and subsequently aid in the development of enzyme reactors that can control the enzyme activities.     

Exosomes as new vesicular lipid transporters involved in cell–cell communication and various pathophysiologies

Exosomes are nanovesicles that have emerged as a new intercellular communication system between an intracellular compartment of a donor cell towards the periphery or an internal compartment of a recipient cell. The bioactivity of exosomes resides not only in their protein and RNA contents but also in their lipidic molecules. Exosomes display original lipids organized in a bilayer membrane and along with the lipid carriers such as fatty acid binding proteins that they contain, exosomes transport bioactive lipids. Exosomes can vectorize lipids such as eicosanoids, fatty acids, and cholesterol, and their lipid composition can be modified by in-vitro manipulation. They also contain lipid related enzymes so that they can constitute an autonomous unit of production of various bioactive lipids. Exosomes can circulate between proximal or distal cells and their fate can be regulated in part by lipidic molecules. Compared to their parental cells, exosomes are enriched in cholesterol and sphingomyelin and their accumulation in cells might modulate recipient cell homeostasis. Exosome release from cells appears to be a general biological process. They have been reported in all biological fluids from which they can be recovered and can be monitors of specific pathophysiological situations. Thus, the lipid content of circulating exosomes could be useful biomarkers of lipid related diseases. Since the first lipid analysis of exosomes ten years ago detailed knowledge of exosomal lipids has accumulated. The role of lipids in exosome fate and bioactivity and how they constitute an additional lipid transport system are considered in this review.     

Significance of nucleotide sequence alignments: a method for random sequence permutation that preserves dinucleotide and codon usage

The similarity of two nucleotide sequences is often expressed in terms of evolutionary distance, a measure of the amount of change needed to transform one sequence into the other. Given two sequences with a small distance between them, can their similarity be explained by their base composition alone? The nucleotide order of these sequences contributes to their similarity if the distance is much smaller than their average permutation distance, which is obtained by calculating the distances for many random permutations of these sequences. To determine whether their similarity can be explained by their dinucleotide and codon usage, random sequences must be chosen from the set of permuted sequences that preserve dinucleotide and codon usage. The problem of choosing random dinucleotide and codon-preserving permutations can be expressed in the language of graph theory as the problem of generating random Eulerian walks on a directed multigraph. An efficient algorithm for generating such walks is described. This algorithm can be used to choose random sequence permutations that preserve (1) dinucleotide usage, (2) dinucleotide and trinucleotide usage, or (3) dinucleotide and codon usage. For example, the similarity of two 60-nucleotide DNA segments from the human beta-1 interferon gene (nucleotides 196-255 and 499-558) is not just the result of their nonrandom dinucleotide and codon usage.      

Preparation and characterization of well defined d-erythro sphingomyelins

A simple semisynthetic procedure for the preparation of various d-erythro sphingomyelins (SPMs), differing in their acyl chains, is described. They were prepared by one-step condensation of the desired free fatty acid with sphingosyl phosphorylcholine (SPC) using dicyclohexylcarbodiimide. The d-erythro SPMs were obtained in high purity, high yields and resemble bovine brain SPM in their chromatographic behavior, infrared, circular dichroism (CD) and proton NMR (PMR) spectra as well as in their rate of hydrolysis by Staphylococcus aureus sphingomyelinase. Multilamellar vesicles can be prepared from the semisynthetic SPMs. Their thermotropic behavior is dependent mainly on the acyl chain though it is also affected by the heterogeneity of the sphingosine base composition. Intact sealed small unilamellar vesicles (SUV) cannot be prepared from a single semisynthetic saturated SPM but can be prepared from their mixtures. This acylation procedure can also be applied for preparing simple neutral glycosphingolipids. The sphingolipids prepared by this method can be used to study metabolism, enzymology and physicochemical properties of d-erythro well defined simple sphingolipids.     

Large scale expression and purification of recombinant RNA in Escherichia coli

Stable, folded RNA are involved in many key cellular processes and can be used as tools for biological, pharmacological and/or molecular design studies. However, their widespread use has been somewhat limited by their fragile nature and by the difficulties associated with their production on a large scale, which were limited to in vitro methods. This work reviews the novel techniques recently developed that allow efficient expression of recombinant RNA in vivo in Escherichia coli. Based on the extensive data available on the genetic and metabolic mechanisms of this model organism, conditions for optimal production can be derived. Combined with a large repertoire of RNA motifs which can be assembled by recombinant DNA techniques, this opens the way to the modular design of RNA molecules with novel properties.     

Identification of individual steroids in biological matrices by mass-analyzed ion kinetic energy spectrometry.

Individual steroids can be distinguished by their mass-analyzed ion kinetic energy (MIKE) spectra. These spectra are taken for the protonated compounds generated by chemical ionization and they yield characteristic fragmentation patterns for different steroids. Isomeric steroids can be differentiated by their MIKE spectra. In some cases this can also be achieved by examining a single ionic reaction rather than the entire spectrum. For example, protonated dehydroepiandrosterone undergoes spontaneous elimination of water while its isomer, testosterone undergoes this reaction with a lower cross-section and then only when excited by collision with target gas. Mixtures of steroids can be analyzed qualitatively. This is illustrated by the analysis of birth control pills (Ovral) for ethynylestradiol and norgestrol. Analysis of urine specimens for particular steroids without pretreatment or preconcentration is possible in some cases.     

Advanced methods for bioreactor characterization.

Bioreactors are characterized by the transport capacities they provide to optimally supply the microorganisms during production process. The transport is performed by flows induced in their cultivation media. In order to understand the extremely complex mixing, mass and heat transfer phenomena encountered, and to perceive their influences on bioreactor performance, sophisticated measuring techniques are required. This review compiles the developments currently in progress to surmount today's shortage of reliable measuring techniques. Measuring techniques are distinguished which can be used on different scales and their application spectra are illustrated by recently obtained results. Several new measuring techniques, which can be employed to resolve the flow structures, are discussed in detail. Only those techniques are considered which can be used to advantage during real cultivations in industrial-scale reactors.     

A Rapid Method for Macerating Phloem

Sieve cells and sieve tube members can be macerated from the phloem of various organs of woody and herbaceous species by au-toclaving the tissue in a mild macerating medium. This treatment does not digest the primary walls or the callose deposits on the sieve areas and sieve plates of the sieve elements. These cells can then be recognized by the fluorescence of their callose after staining with aniline blue. Sometimes adjacent sieve elements fail to separate and one can observe details of their junctures.     

Plant-mediated effects in insect-pathogen interactions

Interactions between insect herbivores and their pathogens can be modulated by host plants. Inter- and intraspecific differences in plant chemistry and structure can alter the susceptibility of insects to infection and the production and environmental persistence of pathogens. Whether plants can manipulate insect pathogens to act as "bodyguards" and increase their own fitness remains to be shown. Reduced insect performance owing to poor plant quality can enhance the susceptibility of an insect to disease while these same phytochemicals can also reduce the effectiveness of entomopathogens in killing the host. As we discuss here, plants have an important role in the evolution of insect-pathogen relationships and a tritrophic perspective should thus be incorporated into the study of insects and their pathogens.     

Biology with information and meaning

It is shown that information and meaning can be defined by operative procedures, and that we need to recognize them as new types of natural entities. They are not quantities (neither fundamental nor derived) because they cannot be measured, and they are not qualities because they are not subjective features. Here it is proposed to call them nominable entities, i.e., entities which can be specified only by naming their components in their natural order.     

Latent phosphorylase phosphatases from rat liver: relationship with the heat-stable inhibitory protein

A high-speed supernatant from rat liver contains at least two latent phosphorylase phosphatases the activities of which are revealed by treatment with ethanol, urea, mercaptoethanol or trypsin. This fraction also contains at least one protein which, after heating, inhibits to various degrees the activated form(s) of the two phosphatases. The two latent enzymes can be separated by cellulase-phosphate chromatography and can be differentiated by their preferential activation by ethanol or trypsin and by their different sensitivity to the inhibitory protein after ethanol activation. Activation of the latent phosphorylase phosphatases by ethanol, urea or mercaptoethanol is not accompanied by the destruction of the precursor of the inhibitory protein whereas activation by trypsin is. However, trypsin treatment of fractions previously activated by ethanol decreases their activity and also increases their sensitivity to the inhibitory protein in a way which is unrelated to the destruction of this inhibitor. Furthermore, some protein fractions, almost free of the precursor of the inhibitory protein can be readily activated by trypsin. In is concluded that the activation of the latent phosphorylase phosphorylase phosphatases is unrelated to the destruction of the inhibitory protein.     

Effect of vibration on the structure and properties of polymer membranes

The structural memory effect in a wave field has been studied by IR spectroscopy using butadiene-styrene and acrylic latex blend films as polymer membrane models. Notable is the enhancement of interactions between phases in such systems, which can cause changes in their local and translational mobility. The response of disperse polymer systems and their composites to nonlinear vibrations means that their deformation properties are affected by vibration, by analogy with orientation phenomena in solid polymers (where the Rebinder effect can appear), and can be considered as a means for modifying polymers, including the manufacture of nanocomposites, polymer biocarriers, etc.     

礁滩分类以及在岩芯中的识别

百余年来涉及生物礁滩的有关论著,从对具体例证的剖析到时空模式的综述,催生着各种分类方案的出台。生物礁滩纵横展布上显示生态环境的相变,礁一滩灰岩具有非均一性,其岩石学特征能指示其生态群落类型。在岩芯中能识别出与各类礁一滩在成因上密切相关的岩性段时,勾勒礁滩体生态单元序列在一定程度上还是可行的。     

Telomeric recombination induced by dysfunctional telomeres

Telomere maintenance is essential for cellular immortality, and most cancer cells maintain their telomeres through the enzyme telomerase. Telomeres and telomerase represent promising anticancer targets. However, 15% of cancer cells maintain their telomeres through alternative recombination-based mechanisms, and previous analyses showed that recombination-based telomere maintenance can be activated after telomerase inhibition. We determined whether telomeric recombination can also be promoted by telomere dysfunction. We report for the first time that telomeric recombination can be induced in human telomerase-positive cancer cells with dysfunctional telomeres.     

Dispersal in Marine Organisms without a Pelagic Larval Phase

In contrast to marine organisms whose offspring go through an extended planktonic stage, the young of others develop directly into benthic juveniles or into yolky nonfeeding larvae that spend only a few hours in the plankton before settling. Yet, paradoxically, many such species have geographic distributions that are comparable to those with a pelagic dispersal stage. This article reviews some of the ways in which these organisms can expand their distributions: drifting, rafting, hitchhiking, creeping, and hopping. Drifting applies to species in which larvae may be short-lived, but adults can detach or be detached from their benthic substratum and be passively carried to new areas, floating at the water's surface or below it. Many encrusting species and mobile species can spread by rafting, settling on natural or artificial floating substrata which are propelled by wind and currents to new regions. Hitchhiking applies to those attaching to vessels or being carried in ballast water of ships to a distant region in which their offspring can survive. Other marine species extend their distributions by hopping from one island of hard substratum or favorable sedimentary microhabitat to another, while creeping species extend their distributions along shores or shelves where habitats remain similar for long distances.     

Neuromodulators and transmitters in respiratory control

The respiratory control system is influenced by classical neurotransmitters and by neuromodulators. The neuromodulators are neuroactive substances that can be secreted at a distance from their receptors and must diffuse to their site of action. Their function can be nonsynaptic and long lasting, and their effect can be direct or indirect via other neuroactive substances. In the central nervous system, a variable degree of mismatch exists between sites of neuromodulator secretion and reception. The assignment of a natural role for a neuromodulator in respiratory control is strengthened by evidence from a variety of experimental approaches, including localization of receptor sites in respiratory-related areas and evidence for natural binding of neuromodulators to these receptors, neurophysiological and respiratory responses to the neuromodulators and their antagonists, and, finally, modulation of specific respiratory responses by neuromodulator antagonists to document the role of the endogenous modulator in eliciting the original response. Neuromodulators that are considered seriously as natural participants in respiratory control include dopamine in peripheral chemoreception and adenosine, endorphins (including enkephalins), serotonin, and substance P in central respiratory regulation.     

甲基营养菌的研究进展

甲基营养菌是一类能够利用一碳化合物作为唯一碳源和能源的微生物,它们在自然界分布广泛.研究表明,甲基营养菌能够直接利用一碳化合物,将其转化成自身代谢的一碳单位,并为生物体提供能源和碳骨架,这组成了一碳代谢的主要部分,它是一种新的代谢体系,可以作为一种新的代谢模式来研究生物代谢和生物进化.本文结合本实验室Methylobacterium sp.MB200的研究情况,主要从分类学、代谢途径、基因组学和应用等方面,论述了甲基营养菌的研究进展.     

Biochemistry of the polyamines

The naturally-occurring polyamines exist in the free form, as N-acetyl derivatives and bound to protein. Their biosynthesis is subject to sensitive control, particularly of ornithine decarboxylase. This enzyme may be multifunctional and a key regulatory protein. Studies, principally with selective inhibitors, have elucidated the roles of polyamines in cell proliferation. Oxidized polyamines, in contrast, can be potent mitotic inhibitors. These effects are reviewed in terms of their chemistry and biochemistry. Their principal distinctions are that they can be made or degraded intracellularly, they can associate electrostatically with macromolecules by means of their spaced cationic groups, and these can be readily converted to covalent bonds.     

A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics

The number of small subunit rRNA sequences is now great enough that the three domains Archaea, Bacteria and Eucarya (Woese et al., 1990) can be reliably defined in terms of their sequence "signatures". Approximately 50 homologous positions (or nucleotide pairs) in the small subunit rRNA characterize and distinguish among the three. In addition, the three can be recognized by a variety of nonhomologous rRNA characters, either individual positions and/or higher-order structural features. The Crenarchaeota and the Euryarchaeota, the two archaeal kingdoms, can also be defined and distinguished by their characteristic compositions at approximately fifteen positions in the small subunit rRNA molecule.