Preparative purification of recombinant thrombolytic protein from complex biological mixtures

The purification of recombinant thrombolytic protein from a crude fermentation broth mixture was examined in antibody exchange (ABx) chromatographic system. ABx system was shown to exhibit unique selectivity for the recombinant protein broth mixture and higher total binding capacity for the recombinant protein than cation-exchange system. The research with the ABx system indicated that these mixed mode interaction systems could be successfully employed in the displacement mode using DEAE-dextran as the displacer.     

SCX separation of recombinant thrombolytic protein from complex biological feeds

The purification of recombinant thrombolytic protein from a crude fermentation broth mixture was examined in strong cation exchange (SCX) Chromatographic system. SCX system was shown to exhibit unique selectivity for the recombinant protein broth mixture. While SCX system showed lower total binding capacity for the recombinant protein than Anti-body Exchange system, it showed sharper separation boundaries in both displacement and step gradient module. The research with the SCX indicated that this separating system could be successfully employed in the displacement mode using DEAE-dextran as the displacer.     

Development of a Plasmid Display System using GAL4 DNA Binding Domain for the <Emphasis Type="Italic">in Vitro</Emphasis> Screening of Functional Proteins

A plasmid display system using GAL4 DNA binding domain (GAL4 DBD) was constructed to enrich the molecular diversity and in vitro selection of functional proteins. Model proteins used were enhanced green fluorescent protein (EGFP) and glutathione S-transferase (GST). The feasibility of this display system was examined using enrichment experiments of target protein from a model protein mixture and identifying the encoding genes by PCR, in which the model protein mixture includes GAL4 DBD/GST fusion protein, GAL4 DBD/EGFP fusion protein, and xylanase. Target proteins of GAL4 DBD/GST and GAL4 DBD/EGFP from the model protein mixture were efficiently isolated by the plasmid display, respectively. The results show that the display system is sufficiently sensitive to select a target protein from a protein mixture, and that it is possible to discover the functional proteins from large libraries using relatively simple approaches.     

Effect of zeolites on protein synthesis in a cell-free system from Escherichia coli

Summary The effects of zeolites on protein synthesis in a cell-free system were investigated. The efficiency of protein synthesis was markedly enhanced upon the addition of zeolites to the reaction mixture. Pretreatment of reaction mixture with the zeolite prior to the start of reaction also stimulated the protein synthesis indicating that the effect is at least partially due to the removal of inhibitory substance(s) from the reaction mixture.     

Assay and structural determination of fructo-oligosaccharides synthesized by an enzymatic system from Penicillium rugulosum

The structure and the fructo-oligosaccharides (FOS) content of a fructan mixture synthesized with an enzymic system from a new strain of rugulosum isolated in our laboratory were investigated by both HPLC and 13 NMR spectrometry. The oligomers were identified as 1-kestose, 1-nystose and 1-fructofuranosyl-nystose. The FOS content of the crude mixture and different commercial products were compared. Results confirmed the interest of such a system for large scale production of FOS.     

ELISA-based method for determining the affinity of bivalent antibodies of two specificities in a mixture

The problem of the evaluation of the affinity for two types of bivalent antibodies in a mixture is considered. It is shown that the binding curve in appropriate coordinates can be used to compose either a system of four equations with four unknowns or a system of two equations with two unknown variables. The numerical solution of these equation systems yields affinity constants for both antibodies and the relationship between concentrations of antibodies studied in the mixture.     

Development of a quantification system of ionic dissociative metabolites using an FT-IR/ATR method

A simultaneous quantification system of ionic dissociative metabolites was developed using a Fourier transform mid-infrared spectroscopic method by focusing our attention on the enzyme reaction from glucose 6-phosphate to fructose 6-phosphate with phosphoglucose isomerase (PGI). We studied the pH dependency of the infrared spectra of the mixture solution for which the PGI reaction was assumed. The infrared spectra of ionic dissociative components in the mixture solution were extracted by multiple linear regression analysis under the assumption of ionic dissociation equilibrium. Additionally, we constructed a simultaneous quantification system using the extracted spectra of the ionic dissociative components on the basis of the ionic dissociation equilibrium. We could accurately estimate the pH value and the concentrations of the ionic dissociative materials in their mixture solution by using this quantification system. In addition, the stability of quantification results for a pK shift was also verified.     

A psychophysical investigation of Beidler's mixture equation

Beidler's mixture equation (1971) describes the relationshipbetween the concentration and composition of a binary mixtureand the magnitude of the neural response. Later this equationwas generalized to a psychophysical level. The purpose of thepresent study is to show that Beidler's mixture equation canbe tested appropriately with indirect psychophysical methods,without the necessity of making assumptions about the magnitudeof the maximum responses to the single compounds which constitutethe mixture. Experiments were carried out using glucose andfructose as tastants. Concentrations of fructose and three equiratiomixture types containing glucose and fructose were matched inperceived sweetness intensities to five different glucose concentrationsusing the method of constant stimuli. The results showed thatBeidler's mixture equation describes accurately the taste interactionbetween glucose and fructose at low sweetness levels. At highsweetness levels the taste system is more efficient, as couldbe expected on the basis of Beidler's mixture equation, becausethe experimentally determined mixture concentrations were lowerthan those predicted by the mixture equation. The findings suggestthat glucose and fructose share common receptors, but that eitherone or both have additional secondary binding mechanisms.     

Solubility of multi-component biodiesel fuel systems

Solubility of biodiesel fuel components in fossil diesel fuel-methanol-rapeseed oil methyl ester, fossil diesel fuel-ethanol-rapeseed oil methyl ester and fossil diesel fuel-ethanol-rapeseed oil ethyl ester systems was investigated. The solubility of components in the fossil diesel fuel-ethanol-rapeseed oil methyl ester system at 20 degrees C was substantially higher than in the fossil diesel fuel-methanol-rapeseed oil methyl ester system. The solubility of components in the fossil diesel fuel-ethanol-rapeseed oil ethyl ester system was slightly lower than in the fossil diesel fuel-ethanol-rapeseed oil methyl ester mixture. The moisture content of ethanol had a great influence on mixture solubility. With decrease of temperature, the solubility of components in the fossil diesel fuel-ethanol-rapeseed oil methyl ester system decreased.     

2 methods of preparing solvent systems for the chromatographic separation of organic acids]

The solvent system of alcohol-acid-water gradually forms an equilibrated amount of ester which facilitates the separation of di- and tri-carboxylic acids on paper chromatograms. Using the system of n-butanol-formic acid-water (3:1:1) as an illustration, two methods of accelerating the system ageing are demonstrated: 1) butanol-acid mixture at a required ratio is kept for several hours before water is added and 2) butanol-acid mixture is passed through the KU-2 layer in the H-form. The effect identical to that achieved as a result of 5-day storage of the solvent system under usual conditions can be reached with the first scheme during 6 hours and with the second scheme during 20-30 min.     

Formulation and Evaluation of Taste Masked Oral Reconstitutable Suspension of Primaquine Phosphate

The purpose of this research was to mask the intensely bitter taste of primaquine phosphate (PRM) and to formulate suspension powder (cachets) of the taste masked drug. Taste masking was done using beta-cyclodextrin. To characterize and formulate taste masked cachets of PRM, the 1:25 M physical mixture was selected based on bitterness score. Phase solubility studies, fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and X-ray powder diffraction (XRPD) were performed to identify the physicochemical interaction between drug and carrier, hence its effect on dissolution. Cachets were evaluated for angle of repose, sedimentation characterization and pH. In vitro drug release studies for physical mixture and kneaded system were performed at pH, 1.2 and 6.8. Bitterness score was evaluated using gustatory sensation test. Phase solubility studies showed weak interaction between PRM and CD. The FTIR, DSC and XRPD studies indicated inclusion complexation in physical mixture and kneaded system. In addition, kneaded system and physical mixture exhibited better drug release at pH 1.2 and negligible effect at pH 6.8. Cachets prepared using physical mixture, (DS24), showed complete bitter taste masking and easy redispersibility. Taste evaluation of cachets in human volunteers rated tasteless with a score of 0 to DS24 and 3 to DS25. Thus, results conclusively demonstrated successful taste masking and formulation of cachets with taste masked drug.     

The methanol-oxidizing system of Methylobacterium extorquens AM1 reconstituted with purified constituents

The electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7 microM methanol dehydrogenase, 3.2 microM cytochrome cH, and 71 nM cytochrome c oxidase (= cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4 microM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.     

A bilayer cell-free protein synthesis system for high-throughput screening of gene products

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.     

Experimental studies of competition between enantiomers in chiral liquid chromatography through their system peaks

Competition between the (+)- and (?) enantiomers of 2,2,2-trifluoro-1-(9-anthryl) ethanol as mobile phase additives was indicated by the chromatographic behavior of their system peaks. Two types of chiral stationary phases were used, one based on dinitrobenzoylphenylglycine and the other on dinitrobenzylphenylethylamine plus tartaric acid. The racemic mixture was used as the mobile phase additive and k′ of their system peaks was studied as a function of the mixture concentration in the mobile phase in both cases. A shift in k′ of the two system peaks was observed and considered as an indication that competition occurred. The areas of the two system peaks were also studied as a function of the concentration of the enantiomers in the samples, using two different compositions of the mobile phase. The dependency of system peaks' area on the sample composition indicated whether competition between the enantiomers occurred. One mobile phase contained 0.1 mM of the racemic mixture, where the area of the two retained system peaks behaved independently, i.e., only the peak corresponding to the enantiomer was affected by its presence in the sample. The other mobile phase contained 0.75 mM of the racemic mixture, and both peaks were affected by the injection of any one of the enantiomers. The interdependency of the system peaks' area on both the enantiomers indicated that their distribution in the chiral system was interrelated due to mutual interactions. A quantitative treatment of the interdependency and competition was excluded, due to the irreversible adsorption of the two enantiomers on the chiral stationary phase after using overloading concentrations. This irreversible adsorption was visualized by the appearance of two retained system peaks of the two residual enantiomers. These system peaks were detected only when the sample contained pure enantiomers due to competition between the enantiomer in the sample with the residual enantiomers in the stationary phase. © 1994 Wiley-Liss, Inc.     

Studies on adrenaline-induced lipolysis in artificial lipid micelles.

Lipid micelles were prepared by incubating a mixture of glycerides (triolein, diolein, and monoolein), and lecithin in Krebs-Ringer phosphate buffer at 37 degrees C for 30 min. It was found that adrenaline stimulated the release of free fatty acids in a lipolytic system consisting of the lipid micelles and adipose tissue lipase. Adrenaline did not increase the cyclic AMP content of the reaction mixture. Dibutyryl cyclic AMP, theophylline, and phospholipase C increased the rate of lipolysis in the system but cyclic AMP and phospholipase D did not.     

Analytical processing of binary mixture information by olfactory bulb glomeruli

Odors are rarely composed of a single compound, but rather contain a large and complex variety of chemical components. Often, these mixtures are perceived as having unique qualities that can be quite different than the combination of their components. In many cases, a majority of the components of a mixture cannot be individually identified. This synthetic processing of odor information suggests that individual component representations of the mixture must interact somewhere along the olfactory pathway. The anatomical nature of sensory neuron input into segregated glomeruli with the bulb suggests that initial input of odor information into the bulb is analytic. However, a large network of interneurons within the olfactory bulb could allow for mixture interactions via mechanisms such as lateral inhibition. Currently in mammals, it is unclear if postsynaptic mitral/tufted cell glomerular mixture responses reflect the analytical mixture input, or provide the initial basis for synthetic processing with the olfactory system. To address this, olfactory bulb glomerular binary mixture representations were compared to representations of each component using transgenic mice expressing the calcium indicator G-CaMP2 in olfactory bulb mitral/tufted cells. Overall, dorsal surface mixture representations showed little mixture interaction and often appeared as a simple combination of the component representations. Based on this, it is concluded that dorsal surface glomerular mixture representations remain largely analytical with nearly all component information preserved.     

Drought conditions alter litter decomposition and nutrient release of litter types in an agroforestry system of China

Evaluating how decomposition rates and litter nutrient release of different litter types respond to changes in water conditions is crucial for understanding global carbon and nutrient cycling. However, it is unclear how decreasing water affects litter mixture interactions for the maize–poplar system in arid regions. Here, the responses of the litter decomposition process and litter mixture interactions in the agroforestry system to changes in water conditions (control, light drought, and moderate drought) were tested. Moderate drought significantly decreased the decomposition rate for poplar leaf and mixed litters, and decomposition rate was significantly reduced for maize straw litter in light and moderate drought stress. The mass loss rates of maize straw and mixed litters were significantly higher than that of the poplar leaf litter under drought conditions, but there was no significant difference among the three litter types in the control. There was no interaction between mass loss of the mixed litter in the control and light drought conditions, and the litter mixture interaction showed nonadditive synergistic interactions under moderate drought. In terms of nutrient release, there was also no interaction between litter mixture with nitrogen and carbon, but there was antagonistic interaction with potassium release under the light drought condition. Our results demonstrate that drought conditions can lead to decreasing decomposition rate and strong changes in the litter mixture interactions from additive effects to nonadditive synergistic effects in moderate drought. Moreover, light drought changed the mixture interaction from an additive effect to an antagonistic interaction for potassium release.     

Bacterial biota of shrimp intestine is significantly modified by the use of a probiotic mixture: a high throughput sequencing approach

The use of probiotics is a common practice of current shrimp aquaculture. Despite the immunophysiological responses that have been measured in shrimp exposed to probiotics, no information is currently available on the effect of this practice on the intestinal microbiota. The objective of this work was to evaluate the effect of a probiotic mixture on the intestinal microbiota of shrimp cultured under farm conditions. A culture-independent method based on high-throughput-sequencing (16S rRNA) was used to examine intestinal bacterial communities. A traditional system (without probiotics) was used as the reference. Targeted metagenomics analysis revealed that the probiotic mixture was based on bacteria in the phyla Proteobacteria and Firmicutes. A total of 23 species of bacteria were detected in the probiotic mixture; of these, 11 were detected in the intestine of shrimp reared in both systems, and 12 were novel for the system. Eight of the novel species were detected in shrimp cultured with the probiotic mixture; however, none of these novel species were related to marine or inclusively aquacultural environments, and only one (Bacillus subtilis) was recognized as probiotic for shrimp. The use of the probiotic mixture modified the bacterial profile of the shrimp intestine; however, most of the bacteria incorporated into the intestine were nonindigenous to the marine environment with no previous evidence of probiotic effects on any marine organism. The use of this probiotic mixture may represent a risk of causing environmental imbalances, particularly because farms using these types of probiotic mixtures discharge their effluents directly into the ocean without prior treatment.     

Model of CE enantioseparation systems with a mixture of chiral selectors. Part II. Determination of thermodynamic parameters of the interconversion in chiral and achiral environments separately

The theoretical assumption that a multi-CS enantioseparation system, the model of which was described in Part I of this work, can be treated as a separation system with only one CS is confirmed by a set of experiments. The model assumes that each individual analyte-CS interaction is fast, fully independent on other interactions and the analyte: CS ratio is 1:1 and that the analyte is present in its concentration small enough not to considerably change the concentration of free CSs. An enantioselective environment in affinity capillary electrophoresis is created using a commercially available mixture of highly sulfated beta-cyclodextrines as chiral selectors (CSs) and lorazepam as an analyte that undergoes interconversion during the separation process. Dependencies of the electrophoretic mobilities of the two enantiomers on concentration of the CSs mixture are proved to follow the proposed multi-CS model. Global rate constants of interconversion are determined at various temperatures and concentrations of the CSs mixture. In accord with the proposed theory, linear dependencies of the global rate constants on CSs concentration are achieved. Intercepts and slopes of these plots correspond to local rate constants of interconversion in achiral (without the mixture of CSs in background electrolyte (BGE)) and chiral (with the CSs mixture in BGE) environments, respectively. The experimentally obtained electropherograms show an excellent fit with those resulting from the computer simulation based on our model.     

Complex mixture discrimination and the role of contaminants

Rats were trained in a 2-alternative odor choice task to discriminate between a 10-component odor mixture and the same mixture with one component removed and replaced with 1 of 3 concentrations of a different monomolecular odor (contaminant). All stimuli were presented within a training session, thus the rat essentially had to learn to discriminate the 10-component mixture from "not" the 10-component mixture. Rats performed most poorly discriminating the complete mixture from the mixture with one component removed and no contaminant added. As the concentration of the contaminant increased from 10 ppm to a concentration equal to the other components (100 ppm), discrimination improved linearly. In analyses of individual differences, rats that spent more time in the sampling port (sampling and making a decision) were more accurate than rats that spent less time. Together, these results emphasize the balance between perceptual stability and perceptual discrimination expressed by the olfactory system dealing with dynamic mixtures and the robust effects of contamination on those processes. In addition, they provide further support that modification of sampling/decision time is a strategy used by rats to deal with difficult discriminations of complex odors.