Carbohydrates: a limit on bacterial diversity within the colon

The human large intestine is recognised as a physiologically important organ responsible for the conservation of water and salts. Through its resident bacteria, it is also capable of complex, enzyme catalysed, hydrolytic-digestive functions that have a high biological impact on the host. These microorganisms metabolise dietary components, principally complex carbohydrates that are not hydrolysed or absorbed in the upper gastrointestinal tract, and in this way, sequester energy for the host, through fermentation. This process involves a series of anaerobic, energy-yielding, catabolic reactions which complete digestive processes in the gut, resulting in end products that in turn influence the distribution of microbial species present as well as having some systemic effects. Some of the bacteria are thought to possess important health-promoting activities, especially with respect to their influence on mucosal and systemic immune responses to disease. These bioactivities can be modulated by substrates that support and influence microbial development, growth and survival. For these reasons, it is necessary to review dietary factors that may delimit bacterial diversity, to be able to predict responses and sensitivities to various environmental pressures and manipulations that occur in this area of human microbiology.     

Elasto-mechanical properties of living cells

The possibility to directly measure the elasticity of living cell has emerged only in the last few decades. In the present study the elastic properties of two cell lines were followed. Both types are widely used as cell barrier models (e.g. blood-brain barrier). During time resolved measurement of the living cell elasticity a continuous quasi-periodic oscillation of the elastic modulus was observed. Fast Fourier transformation of the signals revealed that a very limited number of three to five Fourier terms fitted the signal in the case of human cerebral endothelial cells. In the case of canine kidney epithelial cells more than 8 Fourier terms did not result a good fit. Calculating the correlation between nucleus and periphery of the signals revealed a higher correlation factor for the endothelial cells compared to the epithelial cells.     

Dynamics of living and dead bacterial cells within a mixed-species biofilm during toluene degradation in a biotrickling filter

AIMS: To study the accumulation of the bacterial living cells (LC) and dead cells (DC) in a mixed-species biofilm developed in a 3 l biotrickling filter (BTF) challenged with toluene. METHODS AND RESULTS: The bacterial LC and DC within the biofilm developed on polypropylene Pall rings in a toluene-degrading BTF were enumerated as fluoro-microscopic counts during a 62-operating day period using nucleic acid staining and the direct epifluorescence filter technique. The biofilm development could be separated into three distinct phases: (i) cell attachment, (ii) biofilm establishment and (iii) biofilm maturation. The LC were always dominant (>/=72%) in the biofilm during the establishment phase whereas the average LC fraction decreased to 51% of the total cells in the maturation phase. The concentration of LC and DC was observed to level off after 41 days at 1010 cells per ring. The biofilm thickness and the dry weight increased independently of the cell number during the maturation phase. CONCLUSIONS: After the LC reached a maximum concentration in the biofilm, the biofilm proliferation was only characterized by the accumulation of DC and organic matter. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in the present study are of particular relevance for biofilm mathematical modelling and numerical simulations. They will also be useful to estimate the contribution of the living bacteria within the biofilm in bioprocesses.     

Localization and molecular interactions of mitoxantrone within living K562 cells as probed by confocal spectral imaging analysis

Studying mechanisms of drug antitumor action is complicated by the lack of noninvasive methods enabling direct monitoring of the state and interactions of the drugs within intact viable cells. Here we present a confocal spectral imaging (CSI) technique as a method of overcoming this problem. We applied this method to the examination of localization and interactions of mitoxantrone (1, 4-dihydroxy-5, 8-bis-[([2-(2-hydroxyethyl)-amino]ethyl)amino]-9,10-anthracenedione dihydrochloride), a potent antitumor drug, in living K562 cells. A two-dimensional set of fluorescence spectra of mitoxantrone (MITOX) recorded with micron resolution within a drug-treated cell was analyzed to reveal formation of drug-target complexes and to create the maps of their intracellular distribution. The analysis was based on detailed in vitro modeling of drug-target (DNA, RNA, DNA topoisomerase II) interactions and environmental effects affecting drug fluorescence. MITOX exposed to aqueous intracellular environment, MITOX bound to hydrophobic cellular structures, complexes of MITOX with nucleic acids, as well as the naphtoquinoxaline metabolite of MITOX were simultaneously detected and mapped in K562 cells. These states and complexes are known to be immediately related to the antitumor action of the drug. The results obtained present a basis for the subsequent quantitative analysis of concentration and time-dependent accumulation of free and bound MITOX within different compartments of living cancer cells.     

Toward an understanding of biochemical equilibria within living cells

Four types of environmental effects that can affect macromolecular reactions in a living cell are defined: nonspecific intermolecular interactions, side reactions, partitioning between microenvironments, and surface interactions. Methods for investigating these interactions and their influence on target reactions in vitro are reviewed. Methods employed to characterize conformational and association equilibria in vivo are reviewed and difficulties in their interpretation cataloged. It is concluded that, in order to be amenable to unambiguous interpretation, in vivo studies must be complemented by in vitro studies carried out in well-characterized and controllable media designed to contain key elements of selected intracellular microenvironments.     

Dynamic movement of actin-like proteins within bacterial cells

Actin proteins are present in pro- and eukaryotes, and have been shown to perform motor-like functions in eukaryotic cells in a variety of processes. Bacterial actin homologues are essential for cell viability and have been implicated in the formation of rod cell shape, as well as in segregation of plasmids and whole chromosomes. We have generated functional green fluorescent protein fusions of all three Bacillus subtilis actin-like proteins (MreB, Mbl and MreBH), and show that all three proteins form helical filaments underneath the cell membrane, the pattern of which is distinct for each protein. Time-lapse microscopy showed that the filaments are highly dynamic structures. A number of separate filaments of MreB and Mbl continuously move through the cell along helical tracks underneath the cell membrane. The speed of extension of the growing end of filaments is within the range of known actin polymerization (0.1 microm/s), generating a potential poleward or centreward pushing velocity at 0.24 microm/min for MreB or Mbl, respectively. During nutritional downshift and a block in topoisomerase IV activity, the filaments rapidly disintegrated, showing that movement occurs only in growing cells. Contrary to Mbl and MreBH filaments, MreB filaments were generally absent in cells lacking DNA, providing a further distinction between the three orthologues.     

Rapid multichannel microfluorometry for metabolic and spectral studies in single living cells

A multichannel microspectrofluorometer has been developed for operation on two modes, a ‘morphological’ mode for the assay of intracellular fluorochromes (e.g. NAD(P)H) in correlation with topography, and a ‘spectral’ mode for wavelength analysis of natural cell fluorescence. This instrument is based on an electron bombardment silicon camera tube (EBS) operated in conjunction with a multiscaling computer. The total NAD(P)H emission from 2 × 30 μm cell strips can be analysed in real time (32.8 msec frame scan) with a signal-to-noise ratio over 100:1. The metabolic changes in cytoplasmic regions are compared with those in regions comprising cytoplasm + nucleus, where the major contribution may be nuclear (cf earlier studies). The observation of a ‘multilocalized’ and asynchronous metabolic response is facilitated with substrates such as glucose-1-phosphate, associated with a longer lag period before the initiation of fluorescence changes. The latter largely occur in the 440–480 nm region. Fluorescence spectra recorded from intracellular regions are nearly super-posable to the spectrum obtained from NAD(P)H crystals.     

Changes in the spectral properties of a plasma membrane lipid analog during the first seconds of endocytosis in living cells.

N-[5-(5, 7-dimethyl Bodipy)-1-pentanoyl]-D-erythro-sphingosylphosphorylcholine (C5-DMB-SM), a fluorescent analog of sphingomyelin, has been used in a study of the formation of very early endosomes in human skin fibroblasts. This lipid exhibits a shift in its fluorescence emission maximum from green (approximately 515 nm) to red (approximately 620 nm) wavelengths with increasing concentrations in membranes. When cells were incubated with 5 microM C5-DMB-SM at 4 degrees C and washed, only plasma membrane fluorescence (yellow-green) was observed. When these cells were briefly (< or = 1 min) warmed to 37 degrees C to allow internalization to occur, and then incubated with defatted bovine serum albumin (back-exchanged) at 11 degrees C to remove fluorescent lipids from the plasma membrane, C5-DMB-SM was distributed in a punctate pattern throughout the cytoplasm. Interestingly, within the same cell some endosomes exhibited green fluorescence, whereas others emitted red-orange fluorescence. Furthermore, the red-orange endosomes were usually seen at the periphery of the cell, while the green endosomes were more uniformly distributed throughout the cytoplasm. This mixed population of endosomes was seen after internalization times as short as 7 s and was also seen over a wide range of C5-DMB-SM concentrations (1-25 microM). Control experiments established that the variously colored endosomes were not induced by changes in pH, membrane potential, vesicle size, or temperature. Quantitative fluorescence microscopy demonstrated that the apparent concentration of the lipid analog in the red-orange endosomes was severalfold higher than its initial concentration at the plasma membrane, suggesting selective internalization (sorting) of the lipid into a subset of early endosomes. Colocalization studies using C5-DMB-SM and either anti-transferrin receptor antibodies or fluorescently labeled low-density lipoprotein further demonstrated that this subpopulation of endosomes resulted from receptor-mediated endocytosis. We conclude that the spectral properties of C5-DMB-SM can be used to distinguish unique populations of early endosomes from one another and to record dynamic changes in their number and distribution within living cells.     

Mechanical properties of actin stress fibers in living cells

Actin stress fibers (SFs) play an important role in many cellular functions, including morphological stability, adhesion, and motility. Because of their central role in force transmission, it is important to characterize the mechanical properties of SFs. However, most of the existing studies focus on properties of whole cells or of actin filaments isolated outside cells. In this study, we explored the mechanical properties of individual SFs in living endothelial cells by nanoindentation using an atomic force microscope. Our results demonstrate the pivotal role of SF actomyosin contractile level on mechanical properties. In the same SF, decreasing contractile level with 10 μM blebbistatin decreased stiffness, whereas increasing contractile level with 2 nM calyculin A increased stiffness. Incrementally stretching and indenting SFs made it possible to determine stiffness as a function of strain level and demonstrated that SFs have nearly linear stress-stain properties in the baseline state but nonlinear properties at a lower contractile level. The stiffnesses of peripheral and central portions of the same SF, which were nearly the same in the baseline state, became markedly different after contractile level was increased with calyculin A. Because these results pertain to effects of interventions in the same SF in a living cell, they provide important new understanding about cell mechanics.     

Magainin 2 in action: distinct modes of membrane permeabilization in living bacterial and mammalian cells

Interactions of cationic antimicrobial peptides with living bacterial and mammalian cells are little understood, although model membranes have been used extensively to elucidate how peptides permeabilize membranes. In this study, the interaction of F5W-magainin 2 (GIGKWLHSAKKFGKAFVGEIMNS), an equipotent analogue of magainin 2 isolated from the African clawed frog Xenopus laevis, with unfixed Bacillus megaterium and Chinese hamster ovary (CHO)-K1 cells was investigated, using confocal laser scanning microscopy. A small amount of tetramethylrhodamine-labeled F5W-magainin 2 was incorporated into the unlabeled peptide for imaging. The influx of fluorescent markers of various sizes into the cytosol revealed that magainin 2 permeabilized bacterial and mammalian membranes in significantly different ways. The peptide formed pores with a diameter of ∼2.8 nm (< 6.6 nm) in B. megaterium, and translocated into the cytosol. In contrast, the peptide significantly perturbed the membrane of CHO-K1 cells, permitting the entry of a large molecule (diameter, >23 nm) into the cytosol, accompanied by membrane budding and lipid flip-flop, mainly accumulating in mitochondria and nuclei. Adenosine triphosphate and negatively charged glycosaminoglycans were little involved in the magainin-induced permeabilization of membranes in CHO-K1 cells. Furthermore, the susceptibility of CHO-K1 cells to magainin was found to be similar to that of erythrocytes. Thus, the distinct membrane-permeabilizing processes of magainin 2 in bacterial and mammalian cells were, to the best of our knowledge, visualized and characterized in detail for the first time.     

Effect of anti-ER antibodies within the ER lumen of living cells

We describe the production and partial characterization of 12 monoclonal antibodies raised against a preparation of endoplasmic reticulum membranes obtained from Xenopus laevis liver. Four of the antibodies cross-react with liver melanocytes; two of the antibodies recognize extracellular antigens, whilst the remaining six recognize antigens present in hepatocytes. The concentrations of these latter antigens increase markedly in livers stimulated by estrogen. Western blotting analysis revealed that the six anti-hepatocyte monoclonal antibodies recognize at least five different antigens whose molecular weights are 14K, 18K, 19K, 43K, and 125K. The possible functional involvement of the various antigens in the secretory pathway was investigated using Xenopus oocytes as a surrogate secretory system. The mRNAs coding for the monoclonal antibodies were injected into oocytes and the resulting immunoglobulin chains were translated and assembled into active anti-ER antibodies inside the lumen of the ER. The effect on secretion was then observed. Our data indicate that the binding of antibodies to most antigens of the endoplasmic reticulum membrane may result in a blockage of secretion.     

光谱多样性在植物多样性监测与评估中的应用

光谱多样性是一种基于植物反射电磁辐射光谱的生物多样性维度, 反映了不同波段光谱反射率在植物种内与种间个体之间的变异程度。由于植物反射光谱特征的差异可以综合地反映植物间生化组分和形态特征的差异, 光谱多样性成为植物多样性监测和评估的重要技术手段。该文介绍了光谱多样性的概念及其生态学意义, 比对了多源、多平台光谱数据各自的技术优势和局限性, 并概述了基于光谱多样性的植物多样性监测和评估方法及其应用, 探讨了光谱多样性整合不同维度生物多样性的能力, 展望了光谱多样性在生物多样性研究中的发展前景。光谱多样性能在多空间尺度服务于植物多样性的监测与评估, 特别是依托基于无人机技术的近地面遥感, 可以实现精细尺度植物多样性的监测与评估, 在生物多样性的保护和管理中具有广阔的应用前景。     

Mechanical properties of single living cells encapsulated in polyelectrolyte matrixes

We have studied the mechanical properties of encapsulated Saccharomyces cerevisiae yeast cells by performing AFM force measurements. Single living cells have been coated through the alternate deposition of oppositely charged polyelectrolyte layers and mechanically trapped into a porous membrane. Coated and uncoated cells in presence/absence of bud scars, i.e. scars resulting from previous budding events, have been investigated. No significant differences between encapsulated and bare cells could be inferred from AFM topographs. On the other hand, investigation on the system elasticity through the acquisition and analysis of force curves allowed us to put in evidence the differences in the mechanical properties between the hybrid cell/polyelectrolyte system and the uncoated cells. Analysis of the curves contact region indicates that the polyelectrolyte coating increases the system rigidity. Quantitative evaluation of the cell rigidity through the Hertz-Sneddon model showed that coated cells are characterized by a Young's modulus higher than the value obtained for uncoated cells and similar to the value observed on the bud scar region of uncoated cells.     

Non-protein-bound iron within bacterial cells and the action of bleomycin

Damage to DNA by bleomycin in vitro is entirely dependent on the presence of an iron salt and molecular oxygen. This same reaction has been used to measure non-protein-bound iron in different bacterial species. Although several of the species examined were insensitive to the toxic effects of bleomycin, they all contained a concentration of non-protein-bound iron which should have been sufficient to allow iron-dependent damage to DNA. Other factors such as permeability or inactivation of the drug must explain their apparent insensitivity.     

Breaking out of the mold: diversity within adult stem cells and their niches

During the past several years, it has become increasingly possible to study adult stem cells in their native territories within tissues. These studies have provided new evidence for the existence of stem cells in the breast, muscle, lung and kidney and have led to a deeper understanding of the best-known stem cells in Drosophila and mice. Tissue stem cells are turning out to be diverse, with varying division rates, lineage lengths, and mechanisms of regulation. In addition, stem cells are now known to engage in a wide variety of interactions with neighboring cells and extracellular matrices, and to respond to various neural and hormonal signals. Stem cell niches are also diverse, sometimes harboring multiple stem cell types. Internally, a stem cell's chromatin and cytoskeletal organization play key roles. Understanding how stem cells and their progeny are controlled will illuminate fundamental biological mechanisms that govern the construction and maintenance of tissues within metazoan animals.