Electronic structure calculations as a tool for investigating acyl migrations in ester saponins

The possibility of acyl migrations in ester saponins from Maesa lanceolata was investigated by molecular mechanics and electronic structure calculations carried out on the major constituent maesasaponin IV3 (3beta-O-[[alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-galactopyranosyl-(1-->3)]-[beta-D-galactopyranosyl-(1-->2)]-beta-D-gluco-pyranuronyl]-21beta-angeloyloxy-22alpha-propanoyloxy-13beta,28-oxido-olean-16alpha, 28alpha-diol). It was confirmed that acyl migrations could occur in rings D and E of maesasaponins.     

Three-dimensional fluorescence as a tool for investigating the dynamics of dissolved organic matter in the Lake Biwa watershed

Quantitative and qualitative characterizations of dissolved organic matter (DOM) were carried out at the watershed level in central Japan by measuring dissolved organic carbon (DOC) concentration and the three-dimensional excitation–emission matrix (3-D EEM). DOC concentration was low (mean 37 ± 19 µM C) in the upstream waters, whereas, in general, it increased toward the downstream areas (mean 92 ± 47 µM C). Significant variations in DOC concentration were detected among rivers and channels. DOC concentration in the epilimnion of Lake Biwa increased during the summer period and decreased during the winter period. The lake hypolimnion has lower DOC concentration (mean 87 ± 7 µM C) compared with the epilimnion (107 ± 15 µM C). Fulvic acid (FA)-like substances in the DOM were directly characterized by 3-D EEM. The fluorescence peak for upstream DOM was found in regions with longer wavelengths (excitation/emission 386 ± 6/476 ± 5 nm) compared with downstream and lake DOM (351 ± 12/446 ± 15 nm and 341 ± 6/434 ± 6 nm, respectively). The DOC concentration is correlated with fluorescence peak intensity of FA-like substances in DOM in river waters. Such a relationship was not found in lake DOM. A blueshift of the fluorescence peak from upstream to lake DOM was observed. A decrease in fluorescence intensities was also detected during the summer period. These results may suggest that the degradation of FA-like substances in DOM occurs from natural solar irradiation. Protein-like fluorescence was significantly detected in the lake epilimnion during the summer period. A linear relationship between DOC concentration and protein-like fluorescence indicated that an autochthonous input of DOM gave rise to the increase in DOC concentration in the lake epilimnion during the summer. These results may suggest that the 3-D EEM can be used as a tool for the investigation of DOM dynamics at the watershed level with concurrent measurement of DOC concentration and the fluorescence properties of fulvic acid-like and protein-like substances.     

Mathematical modeling as a tool for investigating cell cycle control networks

Although not a traditional experimental "method," mathematical modeling can provide a powerful approach for investigating complex cell signaling networks, such as those that regulate the eukaryotic cell division cycle. We describe here one modeling approach based on expressing the rates of biochemical reactions in terms of nonlinear ordinary differential equations. We discuss the steps and challenges in assigning numerical values to model parameters and the importance of experimental testing of a mathematical model. We illustrate this approach throughout with the simple and well-characterized example of mitotic cell cycles in frog egg extracts. To facilitate new modeling efforts, we describe several publicly available modeling environments, each with a collection of integrated programs for mathematical modeling. This review is intended to justify the place of mathematical modeling as a standard method for studying molecular regulatory networks and to guide the non-expert to initiate modeling projects in order to gain a systems-level perspective for complex control systems.     

Quantifying changes in gap junction structure as a function of lens fiber cell differentiation

The ocular lens is an ideal model system for studying gap junction structure-function relationships. Here we apply novel methods to quantitatively compare connexin expression over macroscopic distances while simultaneously resolving the intracellular distribution of gap junctions in sub-micron detail. Our approach has identified three distinct zones of connexin density and allowed changes in gap junction plaque size, number and dispersion to be quantified. Our analysis is the first to precisely correlate changes in gap junction plaque structure with the reported changes in gap junction function that occur as a consequence of fiber cell differentiation.     

The fluorescence induction kinetics as a non-destructive tool for investigating spruce treated with ozone

Summary The scattering coefficient of yellow spruce needles exceeds that of green needles by a factor of 2, whereas the fluorescence efficiency is approximately equal for both needle colours. As shown by the angular distribution the fluorescence light is diffusely emitted. However, the scattered light consists of a diffuse and a reflecting portion below 20° with a ratio of the intensities of 1 : 2 at perpendicular observation (0°). Control measurements show that in the rejection region the effective transmission of cut-off-filters commonly used to separate fluorescence light and excitation light exceeds the value calculated from the filter specifications by a factor of 100. Therefore, the portion of the scattered light in the measuring signal must be controlled if the fluorescence induction kinetics is measured from specimen of different colour. A device for the determination of the fluorescence induction kinetics is described which employs a He-Ne laser, a mechanically working shutter with an opening time of 4 ms for the excitation, and a computer for data storage and device control. Two filters select the fluorescence components at 685 nm and 730 nm and they reduce the portion of the scattered light in the measuring signal to 0.18% and 0.55%, respectively. In order to consider the temporal development of the fluorescence kinetics the sampling rate is reduced from 2 kHz to 1 Hz. From the data stored in the computer maximum valueF _P, and steady-state-valueF _S are determined for both fluorescence components. Measurements on 4-year-old spruce exposed to ozone-concentrations of 0, 300 ppb, 600 ppb, and 1000 ppb were repeated every week. With increasing concentration and duration of treatmentR _fd =(F _P-F_s)/F _S was decreased for both fluorescence components. With the highest ozone concentration a reduction ofR _fd of 23% and 24%, respectively, was obtained for the two fluorescence components after three weeks.     

siRNA as a tool for investigating organogenesis: The pitfalls and the promises

Removing the function of a specific gene from a developing organ, by making a ‘knockout’ mouse, is a powerful method for analyzing the molecular pathways that control organogenesis. The technique is expensive, though, in terms of time and money, and complex strategies for producing conditional knockouts are needed for genes that are essential for early development of the embryo, for which an unconditional knockout would be lethal before the organ of interest begins to form. Small interfering RNAs (siRNAs) offer a method of knocking down the expression of specific genes with no need for genomic manipulation. Almost as soon as they had been discovered, siRNAs began to be used to explore the molecular biology of mammalian cells in conventional, two-dimensional culture. They have now also been applied successfully, by several groups, to knock down specific genes in various organ rudiments developing in organ culture. This article reviews the basic technique of siRNA-mediated gene knockdown and how it is being applied to organ culture. It also reviews some of the current problems and challenges in the field, and the ways in which these problems are likely to be overcome.Key words: siRNA, RNAi, organ culture, organogenesis, organ development, 3D culture     

Free nonimmobilized ligands as a tool for purification of proteins

Purification of proteins on a large scale is a complex multistep process, and alternative economic strategies are required. This study presents a novel approach (Affinity Sinking, AS) for purification of native proteins utilizing nonimmobilized modified ligands. The nonimmobilized state of the ligand circumvents the need for immobilizing ligands to polymeric supports. Therefore, purification from large volumes can be accomplished without the use of industrial-scale affinity columns. The mechanism of product capture is formation and precipitation of a specific [target-protein/modified-ligand] complex by using a soluble interconnecting entity that generates an insoluble [target-protein/modified-ligand/interconnecting entity] sediment containing the target protein. Rabbit IgG and two glycoproteins were purified accordingly, utilizing free avidin (as the interconnecting entity) and either desthiobiotinylated-protein A (DB-ProA) or desthiobiotinylated-concanavalin A (DB-ConA) as the modified ligand. The recovery yields for the IgG and the two glycoproteins were 80-86% and 70-75%, respectively. Target proteins are eluted from the generated pellet nearly without disrupting the [modified-ligand/interconnecting entity] macro-complex, thus enabling a practical procedure of recovering target proteins. Leaching of the DB-ProA ligand under eluting conditions (pH 3) was found to be lower than 1%. The two modified ligands, DB-ProA and DB-ConA, were regenerated without any chromatographic procedure in 80% and 85%-89% yield, respectively. The advantage of excluding the polymeric component from the purification process and obtaining highly purified proteins has been demonstrated, and it implies that other contaminants (e.g. endotoxins, prions, host DNA) could be excluded as well, thereby reducing the number of purification steps in a typical downstream process.     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

Alloxan-induced luminol luminescence as a tool for investigating mechanisms of radical-mediated diabetogenicity.

It is not clear whether the muscle wasting commonly observed in hyperthyroidism is due to alteration in the rate of protein synthesis or degradation. The effect of experimental hyperthyroidism on skeletal-muscle proteolysis in the rat was studied by measuring alanine and tyrosine release from isolated skeletal muscles in vitro and 3-methyl-histidine excretion in vivo. Alanine release from the isolated epitrochlaris-muscle preparation was increased as soon as 24h after a 25 microgram dose of L-tri-iodothyronine in vivo. Conversely, alanine release from muscles of hypothyroid rats was decreased, but restored by L-tri-iodothyronine supplementation before death. Furthermore, 3-methylhistidine excretion was increased in hyperthyroid rats throughout an 18-day treatment period. The increased amino acid release from isolated muscles and the increased 3-methylhistidine excretion in vivo strongly suggests that hyperthyroidism increases skeletal-muscle proteolysis. Furthermore, the thyroid-hormone concentration may be an important factor in regulating muscle proteolysis.     

Additive partitioning as a tool for investigating the flora diversity in oceanic archipelagos

This paper introduces the integration of additive partitioning with species—area relationships to island biogeography in order to address the question “How are the pteridophyte and spermatophyte native and endemic flora of different oceanic archipelagos partitioned across islands?”.Species richness data of all endemic species and all native species of pteridophytes and spermatophytes were obtained for the Azores, Canaries and Cape Verde in the Atlantic Ocean and Galápagos, Hawaii and Marquesas in the Pacific Ocean. Additive partitioning of species diversity was used to quantify how much of the total diversity of an oceanic archipelago flora (γ-diversity) is due to (i) the mean species richness of the flora of each island (α-diversity), (ii) the variability in species richness of the floras across islands (β_Nestedness) and (iii) the complementarity in species composition of the floras of different islands (β_Replacement). The analysis was separately performed for the native and endemic pteridophyte and spermatophyte floras.The diversity partitioning of the six archipelagos showed large differences in how the flora of each archipelago is partitioned among the α, β_Nestedness and β_Replacement components, for pteridophytes and spermatophytes and for all endemic species and all native species. The α-diversity was more important for all native species than for endemic species and more important for pteridophytes than for spermatophytes, with the Azores showing outstanding high values of α-diversity. The β_Nestedness was higher for pteridophytes than for spermatophytes and higher for endemic species than for all native species in both pteridophytes and spermatophytes. The values of β_Replacement suggested that: (i) the spermatophyte native flora is more differentiated across islands than the pteridophyte native flora and (ii) the pteridophyte endemic flora and, especially, the spermatophyte endemic flora are more differentiated across islands than the corresponding native flora. An outstanding value of β_Replacement for endemic and all native spermatophytes was found in Hawaii, confirming the biogeographical island differentiation in this archipelago.     

In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

Photosynthesis Research - We report the application of NMR dynamic spectral editing for probing the structure and dynamics of molecular constituents in fresh, intact cells and in freshly prepared...     

Functional magnetic resonance imaging as a tool for investigating human cortical motor function.

Non-invasive functional magnetic resonance imaging (fMRI) mapping techniques sensitive to the local changes of blood flow, blood volume, and blood oxygenation which accompany neuronal activation have been widely used over the last few years to investigate the functional organization of human cortical motor systems, and specifically of the primary motor cortex. Validation studies have demonstrated a good correspondence between quantitative and topographic aspects of data acquired by fMRI and positron emission tomography. The spatial and temporal resolution affordable by fMRI has allowed to achieve new important information on the distributed representation of hand movements in multiple functional modules, and on the intensity and spatial extent of neural activation in the contralateral and ipsilateral primary motor cortex in relation to parametric and nonparametric aspects of movement and to the degree of handedness. Neural populations with different functional characteristics have been identified in anatomically defined regions, and the temporal aspects of the activation during voluntary movement tracked in different components of the motor system. Finally, this technique has proved useful to deepen our understanding of the neural basis of motor imagery, demonstrating increased activity in the primary motor cortex during mental representation of sequential finger movements.     

Molecular dynamics simulations as a tool for improving protein stability

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.     

Continuous culture as a tool for investigating the growth physiology of heterotrophic hyperthermophiles and extreme thermoacidophiles

Although there is great scientific and technological interest in examining the physiology and bioenergetics of microorganisms from extreme environments, difficulties encountered in their cultivation and lack of genetic systems hampers the investigation of these issues. As such, we have adapted methods for continuous cultivation of mesophilic organisms to extremes of temperature and pH to study extremophiles. Since the risk for contamination of extremophilic continuous cultures is relatively small, long-term, steady state experiments investigating physiological response to culture perturbations are possible. Experiments along these lines have provided insights into the significance of specific enzymes in the metabolism of particular substrates, in addition to providing a better understanding of stress response and unusual physiological characteristics of hyperthermophilic and extremely thermoacidophilic microorganisms. Several examples are provided here, including the thermal stress response of Metallosphaera sedula (T(opt) 74 °C) growing at pH 2.0, and the response of the heterotrophic hyperthermophiles Pyrococcus furiosus (T(opt) 98 °C), Thermococcus litoralis (T(opt) 88 °C) and T. maritima (T(opt) 80 °C) to changes in growth medium. Also discussed will be how the same experimental systems have been used to study exopolysaccharide production and biofilm formation by hyperthermophilic heterotrophs and facilitated the estimation of bioenergetic parameters for these organisms under a variety of growth conditions. Continuous culture, used in conjunction with genome sequence information, two-dimensional gel electrophoresis and differential gene expression, can provide important insights into the metabolism of high temperature extremophiles.     

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.     

Connexin phosphorylation as a regulatory event linked to gap junction internalization and degradation

Gap junction proteins, connexins, are dynamic polytopic membrane proteins that exhibit unprecedented short half-lives of only a few hours. Consequently, it is well accepted that in addition to channel gating, gap junctional intercellular communication is regulated by connexin biosynthesis, transport and assembly as well as the formation and removal of gap junctions from the cell surface. At least nine members of the 20-member connexin family are known to be phosphorylated en route or during their assembly into gap junctions. For some connexins, notably Cx43, evidence exists that phosphorylation may trigger its internalization and degradation. In recent years it has become apparent that the mechanisms underlying the regulation of connexin turnover are quite complex with the identification of many connexin binding molecules, a multiplicity of protein kinases that phosphorylate connexins and the involvement of both lysosomal and proteasomal pathways in degrading connexins. This paper will review the evidence that connexin phosphorylation regulates, stimulates or triggers gap junction disassembly, internalization and degradation.     

Tetracysteine Genetic Tags Complexed with Biarsenical Ligands as a Tool for Investigating Gap Junction Structure and Dynamics

Gap junctions (GJ) are defined as contact regions between two adjacent cells containing tens to thousands of closely packed membrane channels. Cells dynamically modulate communication through GJ by regulating the synthesis, transport and turnover of these channels. Previously, we engineered a recombinant connexin43 (Cx43) by genetically appending a small tetracysteine peptide motif containing the sequence -Cys-Cys-Xaa-Xaa-Cys-Cys- to the carboxy terminus of Cx43 (Cx43-TC) (3). Cx43-TC was stably expressed in HeLa cells and was specifically labeled by exposing the cells to membrane-permeant non-fluorescent ligands, such as FlAsH (a fluorescein derivative) and ReAsH (a resorufin derivative). Direct correlation of live cell images with high resolution EM detection was possible because bound ReAsH not only becomes fluorescent, but can also be used to initiate the photoconversion of diaminobenzidine (DAB) that causes the localized polymerization of an insoluble osmiophilic precipitate then visible by EM. Cx43-TC GJ's could be labeled with ReAsH and photooxidized to give selectively stained channels. Here, how the development of these tetracysteine tags complexed with appropriate ligands are useful for experiments spanning resolution ranges from light microscopy to electron tomography to molecular purification and detection is described.