THE RÔLE OF PLANT CELLS IN THE ENSILAGE PROCESS: AN APPROACH TO THE PROBLEM

SUMMARY: An apparatus is described in which sufficient microbe-free timothy grass to make 'silage' on a laboratory scale was grown. In one experiment the chemical changes which occurred during the period of ensiling were measured. They could be attributed only to the metabolism of the plant cells.     

CHEMICAL AND PHYSICAL CHANGES IN GELATIN SOLUTIONS DURING HYDROLYSIS

1. The change in viscosity and the corresponding increase in the carboxyl groups, as determined by the formol titration, has been determined in gelatin solutions during the progress of hydrolysis by pepsin. 2. Very marked changes in viscosity are found to result from very slight chemical changes. If the viscosity is increased by the addition of acid a greater change in viscosity (volume of solute) is caused by the same percentage change in the number of carboxyl groups. The percentage change in the volume of solute, caused by the same percentage increase in the number of carboxyl groups, is independent of the concentration of gelatin. 3. These results are in agreement with the idea that the high viscosity of gelatin solutions is due to the presence of swollen micells, since a slight chemical hydrolysis may be sufficient to rupture a micella and so cause a very large change in viscosity.     

A simplified method for estimating ammonium oxidising bacteria

Summary Most probable number of ammonium oxidising bacteria in soil has been estimated without the need to carry out chemical tests to determine the production of nitrite or nitrate in dilution tubes. The medium used contained phenol red which changes colour from pink to yellow as the oxidation of ammonium to nitrate decreases the pH. This makes it possible to determine growth visually. An incubation period of 8 weeks at 25°C was sufficient to obtain maximum estimates of ammonium oxidising bacteria in the soils tested.     

Indirect Information Transfer: Three‐Spined Sticklebacks Use Visual Alarm Cues From Frightened Conspecifics About an Unseen Predator

Animals can attempt to reduce uncertainty about their environment by gathering information personally or by observing others' interactions with the environment. There are several sensory modalities that can be used to transmit social information from chemical to visual to audible cues. When predation risk is variable, visual cues of conspecific behavior might be especially telling about the presence of a potential threat; however, most studies couple visual and chemical cues together. Here, we tested whether visual behavioral cues from frightened conspecifics were sufficient to indirectly transfer information about the presence of an unseen predator in three‐spined sticklebacks. Our results demonstrate that visual behavioral cues from conspecifics about the presence of a predator are sufficient to induce an antipredator response. This suggests that information transfer can occur rapidly in the absence of chemical cues and that some individuals weigh social information more heavily than others.     

Biochemical effects of thiabendazole and cambendazole on Hymenolepis diminuta (Cestoda) in vivo

An investigation of the chemotherapeutic and biochemical effects of two benzimidazole anthelmintics, thiabendazole (TBZ) and cambendazole (CBZ), on Hymenolepis diminuta in experimentally infected rats is reported. Thiabendazole was active against H. diminuta at a relatively high dosage. A single oral dose of TBZ at 250 mg/kg body weight on day 15 of infection eliminated 100% of the tapeworms as determined at necropsy 5 days after treatment. The chemotherapeutic actions of TBZ on H. diminuta were accompanied by marked changes in worm weight and chemical composition. Tapeworms recovered from rats that had received a therapeutically effective dose of TBZ 24 hr earlier were significantly smaller and contained much less glycogen (as a percent of the wet weight) than worms from unmedicated controls. Protein concentrations increased in TBZ-treated worms and at a rate sufficient to offset the decline in glycogen concentration. Glycogen/protein ratios in TBZ-treated worms were significantly lower than the corresponding control values. Cambendazole proved to be five times more potent than TBZ against H. diminuta and produced the same basic changes in worm weight and chemical composition within 18 hr of treatment of the host. Administration of a single oral dose of TBZ or CBZ to the host produced in H. diminuta another change, the onset of which coincided with, or preceded, the gross alterations in worm weight and chemical composition. That change, observed in in vitro studies carried out 14 hr after treatment, revealed that tapeworms from drug-treated rats absorbed and metabolized much smaller quantities of exogenous glucose than did the controls, and the ability of the worm to accumulate glucose against a concentration difference was significantly depressed.     

Discrimination of toxic impacts of various chemicals using chemical–gene expression profiling of Escherichia coli DNA microarray

The expression levels of 96 genes were characterized and differentiated using a cDNA microarray after the bacterium Escherichia coli was exposed to numerous toxic chemicals. In all, the effects of 14 different chemicals and 1 mixture were investigated using 1-h exposure data to provide information about physiological changes brought on by the stress experienced and interaction of chemical–gene expression. Hierarchical clustering analysis showed that the genes could be sub-grouped based upon their expression patterns while each also showed unique signatures to each chemical tested when examined using a principal component analysis (PCA). By constructing a chemical–gene expression profiling based on changes in the expression of the genes for each chemical, we were able to identify the chemicals effects and gene targets more systematically. Despite the fact that only a small number of genes were used for gene expression analysis, they were sufficient to discriminate between the effects of each exposure. It was found that the use of a single time point for expression analysis was insufficient for interpreting the effects a given chemical has on the bacterium. Such information cannot be obtained from conventional toxicity studies, demonstrating that chemical–gene expression profiling method based on the hierarchical clustering analysis and principal component analysis (PCA) in toxicity monitoring offers a new perspective for bio-monitoring and information on dynamic changes occurring at the sub-cellular level.     

Design and analysis of experiments on mutagenicity : I. Minimal sample sizes

The probability (power) of collecting sufficient data to demonstrate statistically that a chemical is mutagenic is examined. A formula is derived with which one may calculate the likelihood of correctly concluding a chemical is mutagenic based upon (1) the total sample size, (2) the relative size of the experimental group, (3) the spontaneous mutation rate and (4) the minimal increase in mutation rate that one is concerned in detecting. Figures are also presented which allow one to determine the minimal total sample size required to assure oneself a reasonable chance of successfully demonstrating a chemical to be mutagenic.

It is found that the best designed (most powerful) experiments are those with approximately equal numbers of indviduals in the experimental and control groups. Those experiments in which excess numbers of individuals are allotted to either the experimental or control groups suffer a substantial reduction in power. In addition, caution should be exercised when concluding that a chemical is not mutagenic. The inability to collect sufficient data to demonstrate the mutagenicity of a chemical might be attributable to one's using too small a total sample size rather than to lack of mutagenic activity, especially in mutagenicity assay systems involving very small spontaneous mutation rates.     

Alterations in polyamine metabolism during embryonal carcinoma cell differentiation in vitro

Murine embryonal carcinoma (EC) cells can be stimulated to differentiate by several chemical inducers. Since the response of EC cells to induction is likely to occur shortly after exposure to the inducer, we report here the changes that occur in polyamine levels in a number of EC cell lines shortly after exposure to two chemical stimuli, alpha-difluoromethylornithine (DFMO) and retinoic acid (RA). Our results suggest that polyamine levels are important in determining the state of EC cell differentiation, but that reduction in these levels alone is not sufficient to induce differentiation in all the EC cell lines tested. Also, it is apparent that RA does influence levels of polyamines. However, this influence does not seem to be mediated through direct interaction with ODCase.     

Identification of molecular pathologies sufficient to cause neuropathic excitability in primary somatosensory afferents using dynamical systems theory

Pain caused by nerve injury (i.e. neuropathic pain) is associated with development of neuronal hyperexcitability at several points along the pain pathway. Within primary afferents, numerous injury-induced changes have been identified but it remains unclear which molecular changes are necessary and sufficient to explain cellular hyperexcitability. To investigate this, we built computational models that reproduce the switch from a normal spiking pattern characterized by a single spike at the onset of depolarization to a neuropathic one characterized by repetitive spiking throughout depolarization. Parameter changes that were sufficient to switch the spiking pattern also enabled membrane potential oscillations and bursting, suggesting that all three pathological changes are mechanistically linked. Dynamical analysis confirmed this prediction by showing that excitability changes co-develop when the nonlinear mechanism responsible for spike initiation switches from a quasi-separatrix-crossing to a subcritical Hopf bifurcation. This switch stems from biophysical changes that bias competition between oppositely directed fast- and slow-activating conductances operating at subthreshold potentials. Competition between activation and inactivation of a single conductance can be similarly biased with equivalent consequences for excitability. "Bias" can arise from a multitude of molecular changes occurring alone or in combination; in the latter case, changes can add or offset one another. Thus, our results identify pathological change in the nonlinear interaction between processes affecting spike initiation as the critical determinant of how simple injury-induced changes at the molecular level manifest complex excitability changes at the cellular level. We demonstrate that multiple distinct molecular changes are sufficient to produce neuropathic changes in excitability; however, given that nerve injury elicits numerous molecular changes that may be individually sufficient to alter spike initiation, our results argue that no single molecular change is necessary to produce neuropathic excitability. This deeper understanding of degenerate causal relationships has important implications for how we understand and treat neuropathic pain.     

Tor1 regulates protein solubility in Saccharomyces cerevisiae

Accumulation of insoluble protein in cells is associated with aging and aging-related diseases; however, the roles of insoluble protein in these processes are uncertain. The nature and impact of changes to protein solubility during normal aging are less well understood. Using quantitative mass spectrometry, we identify 480 proteins that become insoluble during postmitotic aging in Saccharomyces cerevisiae and show that this ensemble of insoluble proteins is similar to those that accumulate in aging nematodes. SDS-insoluble protein is present exclusively in a nonquiescent subpopulation of postmitotic cells, indicating an asymmetrical distribution of this protein. In addition, we show that nitrogen starvation of young cells is sufficient to cause accumulation of a similar group of insoluble proteins. Although many of the insoluble proteins identified are known to be autophagic substrates, induction of macroautophagy is not required for insoluble protein formation. However, genetic or chemical inhibition of the Tor1 kinase is sufficient to promote accumulation of insoluble protein. We conclude that target of rapamycin complex 1 regulates accumulation of insoluble proteins via mechanisms acting upstream of macroautophagy. Our data indicate that the accumulation of proteins in an SDS-insoluble state in postmitotic cells represents a novel autophagic cargo preparation process that is regulated by the Tor1 kinase.     

Perfusion-induced redox differences in cytochrome c oxidase: ATR/FT-IR spectroscopy

Attenuated total reflection (ATR) spectroscopy brings an added dimension to studies of structural changes of cytochrome c oxidase (CcO) because it enables the recording of reaction-induced infrared difference spectra under a wide variety of controlled conditions (e.g. pH and chemical composition), without relying on light or potentiometric changes to trigger the reaction. We have used the ATR method to record vibrational difference spectra of CcO with reduction induced by flow-exchange of the aqueous buffer. Films of CcO prepared from Rhodobacter sphaeroides and beef heart mitochondria by reconstitution with lipid were adhered to the internal reflection element of the ATR device and retained their full functionality as evidenced by visible spectroscopy and time-resolved vibrational spectroscopy. These results demonstrate that the technique of perfusion-induced Fourier-transform infrared difference spectroscopy can be successfully applied to a large, complex enzyme, such as CcO, with sufficient signal/noise to probe vibrational changes in individual residues of the enzyme under various conditions.     

Calibration of nanowatt isothermal titration calorimeters with overflow reaction vessels

Obtaining accurate results with nanowatt titration calorimeters with overflow cells requires mass calibration of the buret injection volume, chemical calibration of the reaction vessel effective volume, and chemical calibration of the calorimetric factor used to convert the measured electrical signal to heat rate. Potential errors in electrical calibration of power compensation calorimeters require validation of the calorimetric factor with chemical reactions with accurately known stoichiometries and enthalpy changes. The effective volume of the reaction vessel can be determined from the endpoint of a quantitative reaction with known stoichiometries. Methods for calibration and potential calibration errors to be avoided are described. Publication of results obtained must include data on calibrations and sufficient raw data to assess precision and accuracy of the results.     

Adverse Reactions to Antimicrobial Therapy

Adverse reactions to antimicrobial agents consist of toxic effects mediated through chemical changes in tissue cells, sensitivity reactions produced by antigen-antibody reactions, and biological effects caused by alteration of the bacterial flora in the body. Patients vary in their susceptibility to these because of genetically determined differences in enzyme and protein make-up, differences in immunologic reactivity, differences in environment, or because of the effects of disease. Some antibiotics are capable of producing almost invariable damage to certain tissues if given long enough in sufficient dosage, whereas others produce disturbances in only occasional patients. Almost any organ in the body may be involved, indicating that the metabolic processes affected by these agents are represented to a varying degree in the cells of the host. Little is known of the specific mechanisms involved, but as information is acquired, it should be possible to reduce the incidence of reactions.     

Soluble metals in the atmosphere and their biological implications

Multivariate statistical analysis has been applied to time series measurements of aerosol elemental composition from PIXE analysis of filter samples, and principal components have been resolved that represent distinct particle types in an external mixture in the atmosphere. In this study, it is argued that a combination of chemical and statistical analyses of the data may be more powerful in determining chemical species in atmospheric aerosols than studies that employ mainly direct chemical analysis of chemical species in unresolved mixtures of aerosol particle samples. Sulfur is generally associated with mineral dust elements. It is reasoned that the association may represent sulfuric acid coatings on particles that can lead to mineral dissolution and solubilization of significant amounts of aluminum, iron, and other metals. Upon wet or dry deposition to the surface, the fluxes of these metals in biologically-available form may be sufficient to affect primary productivity in the world ocean and cause ecological damage in lakes. As a consequence, the fluxes of biogenic trace gases to the atmosphere may be changed, possibly leading to changes in the tropospheric concentration of ozone. The inputs to lakes of soluble aluminum, which is toxic to fish, may be partly by deposition directly from the atmosphere, thus not limited to leaching of soils by acid deposition. Human inhalation of soluble aluminum and other solulilized mineral metals may account, in part, for the observed geographic pattern of deaths attributed to chronic obstructive pulmonary disease (COPD) that show high rates in cities of the Western US and the southeast region, but low in most of the midwest and northeast.     

Surface pH controls purple-to-blue transition of bacteriorhodopsin. A theoretical model of purple membrane surface.

We have developed a surface model of purple membrane and applied it in an analysis of the purple-to-blue color change of bacteriorhodopsin which is induced by acidification or deionization. The model is based on dissociation and double layer theory and the known membrane structure. We calculated surface pH, ion concentrations, charge density, and potential as a function of bulk pH and concentration of mono- and divalent cations. At low salt concentrations, the surface pH is significantly lower than the bulk pH and it becomes independent of bulk pH in the deionized membrane suspension. Using an experimental acid titration curve for neutral, lipid-depleted membrane, we converted surface pH into absorption values. The calculated bacteriohodopsin color changes for acidification of purple, and titrations of deionized blue membrane with cations or base agree well with experimental results. No chemical binding is required to reproduce the experimental curves. Surface charge and potential changes in acid, base and cation titrations are calculated and their relation to the color change is discussed. Consistent with structural data, 10 primary phosphate and two basic surface groups per bacteriorhodopsin are sufficient to obtain good agreement between all calculated and experimental curves. The results provide a theoretical basis for our earlier conclusion that the purple-to-blue transition must be attributed to surface phenomena and not to cation binding at specific sites in the protein.     

The bhopalator: a molecular model of the living cell based on the concepts of conformons and dissipative structures

A molecular model of the living cell has been formulated based on a new theory of enzymic catalysis which takes into account the complementary roles of free energy and genetic information. The elementary units of free energy and genetic information that are necessary and sufficient for effectuating molecular mechanisms responsible for the life of the cell are called conformons. Conformons are visualized as a collection of a small number of catalytic residues of enzymes or segments of nucleic acids that are arranged in space and time with appropriate force vectors so as to cause chemical transformations or physical changes of a substrate or a bound ligand. So defined, conformons provide a plausible molecular means to link the genetic information stored in DNA and its ultimate expression, namely networks of coupled intracellular biochemical reactions and physical processes maintained by a continuous dissipation of free energy--dissipative structures of Prigogine. The proposed model of the living cell appears to possess the potential for bridging the gap between molecular biology and the biology of multicellular systems.     

Genome scale enzyme-metabolite and drug-target interaction predictions using the signature molecular descriptor

MOTIVATION: Identifying protein enzymatic or pharmacological activities are important areas of research in biology and chemistry. Biological and chemical databases are increasingly being populated with linkages between protein sequences and chemical structures. There is now sufficient information to apply machine-learning techniques to predict interactions between chemicals and proteins at a genome scale. Current machine-learning techniques use as input either protein sequences and structures or chemical information. We propose here a method to infer protein-chemical interactions using heterogeneous input consisting of both protein sequence and chemical information. RESULTS: Our method relies on expressing proteins and chemicals with a common cheminformatics representation. We demonstrate our approach by predicting whether proteins can catalyze reactions not present in training sets. We also predict whether a given drug can bind a target, in the absence of prior binding information for that drug and target. Such predictions cannot be made with current machine-learning techniques requiring binding information for individual reactions or individual targets.     

Modeling of Hidden Structures Using Sparse Chemical Shift Data from NMR Relaxation Dispersion

NMR relaxation dispersion measurements report on conformational changes occurring on the μs-ms timescale. Chemical shift information derived from relaxation dispersion can be used to generate structural models of weakly populated alternative conformational states. Current methods to obtain such models rely on determining the signs of chemical shift changes between the conformational states, which are difficult to obtain in many situations. Here, we use a “sample and select” method to generate relevant structural models of alternative conformations of the C-terminal-associated region of Escherichia coli dihydrofolate reductase (DHFR), using only unsigned chemical shift changes for backbone amides and carbonyls (¹H, ¹⁵N, and ¹³C′). We find that CS-Rosetta sampling with unsigned chemical shift changes generates a diversity of structures that are sufficient to characterize a minor conformational state of the C-terminal region of DHFR. The excited state differs from the ground state by a change in secondary structure, consistent with previous predictions from chemical shift hypersurfaces and validated by the x-ray structure of a partially humanized mutant of E. coli DHFR (N23PP/G51PEKN). The results demonstrate that the combination of fragment modeling with sparse chemical shift data can determine the structure of an alternative conformation of DHFR sampled on the μs-ms timescale. Such methods will be useful for characterizing alternative states, which can potentially be used for in silico drug screening, as well as contributing to understanding the role of minor states in biology and molecular evolution.     

Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae)

The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca(2+)](i) in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.