Methods based on artificial intelligence,for controlling biotechnological processes. Monitoring of the concentration of dissolved oxygen (part III)

A real-time artificial intelligence method for controlling the concentration of dissolved oxygen is proposed. Two projection versions of algorithms are considered in this paper. The versions vary in structure of the intelligent agents. One of the agents represents an automaton of expedient behaviour, the structure of the other consists of two automata which behave expediently in a complicated random medium. The first algorithm holds a check on a trend of change in values. The second version of the algorithm takes into account both the trend of change in the values and the speed of change in the values. Simulation studies show that expedient behaviour of the automata in the random medium for the control of dissolved oxygen concentration can bring about a good performance.     

Determination of the anti‐viral drug Ribavirin in dosage forms via micelle‐enhanced spectrofluorimetric method

A simple and sensitive spectrofluorimetric method was developed for the determination of Ribavirin in pharmaceutical formulations. The proposed method was based on the fluorescence spectral behaviour of Ribavirin in a sodium dodecyl sulfate (SDS) micellar system. In an aqueous acetate buffer solution of pH 4.0, the fluorescence intensity of Ribavirin was significantly enhanced by about 217% in the presence of SDS. Fluorescence intensity was measured at 396 nm after excitation at 270 nm for Ribavirin. The fluorescence‐concentration plot was rectilinear over the range of 0.01‐3.0 µg/mL for Ribavirin with a lower detection limit of 5.02 x 10^‐3 µg/mL. The method was successfully applied to the analysis of the drug in its commercial capsules. Results were in good agreement with those obtained with the official method. The application of the proposed method was extended to stability studies of Ribavirin after exposure to different forced degradation conditions such as acidic, alkaline, photo and oxidative conditions according to ICH guidelines. Copyright © 2012 John Wiley & Sons, Ltd.     

Crystal structure and functional analysis of Escherichia coli glutamate decarboxylase

Glutamate decarboxylase is a vitamin B6-dependent enzyme, which catalyses the decarboxylation of glutamate to gamma-aminobutyrate. In Escherichia coli, expression of glutamate decarboxylase (GadB), a 330 kDa hexamer, is induced to maintain the physiological pH under acidic conditions, like those of the passage through the stomach en route to the intestine. GadB, together with the antiporter GadC, constitutes the gad acid resistance system, which confers the ability for bacterial survival for at least 2 h in a strongly acidic environment. GadB undergoes a pH-dependent conformational change and exhibits an activity optimum at low pH. We determined the crystal structures of GadB at acidic and neutral pH. They reveal the molecular details of the conformational change and the structural basis for the acidic pH optimum. We demonstrate that the enzyme is localized exclusively in the cytoplasm at neutral pH, but is recruited to the membrane when the pH falls. We show by structure-based site-directed mutagenesis that the triple helix bundle formed by the N-termini of the protein at acidic pH is the major determinant for this behaviour.     

Some observations on the periodate oxidation of amino compounds

Various aliphatic and aromatic amines are oxidized by sodium metaperiodate and these reactions have been studied quantitatively in acidic, unbuffered and basic media. Significant differences have been observed between the behaviour of aliphatic and aromatic amines. Certain compounds also behaved differently under acidic and basic conditions. These reactions are related to the periodate oxidation of amino acids and, from observations on a number of glycine derivatives, a reaction mechanism is proposed for this process.     

Extractive fermentation for lactic acid production

Lactic acid extractive fermentation was demonstrated using Alamine 336 in oleyl alcohol at acidic pH. The use of an efficient extraction system was possible through employment of the cell immobilization procedure. Process modeling was performed to relate the various process parameters such as flow rate, concentration, and pH. In experiments with 15% Alamine 336/oleyl alcohol, the bioreactor operation resulted in a higher productivity (12 g/L gel h) compared to that of a control fermentation (7 g/L gel h). Strategies for optimizing the extractive fermentation process were proposed considering both productivity and product recovery.     

Stability‐indicating micelle‐enhanced spectrofluorimetric method for determination of loratadine and desloratadine in dosage forms

A highly sensitive and simple spectrofluorimetric method was developed for the determination of loratadine (LRT) and desloratadine (DSL) in their pharmaceutical formulations. The proposed method is based on investigation of the fluorescence spectral behaviour of LRT and DSL in a sodium dodecyl sulphate (SDS) micellar system. In aqueous solution of acetate buffer of pH 4.5, the fluorescence intensities of both LRT and DSL were greatly enhanced (240%) in the presence of SDS. The fluorescence intensity was measured at 438 nm after excitation at 290 nm for both drugs. The fluorescence–concentration plots were rectilinear over the range 0.05–2.0 µg/mL for both LRT and DSL, with lower detection limits of 5.13 × 10^?3 and 6.35 × 10^?3 µg/mL for LRT and DSL, respectively. The method was successfully applied to the analysis of the two drugs in their commercial tablets, capsules and syrups, and the results were in good agreement with those obtained with the official or comparison methods. The proposed method is specific for the determination of LRT in the presence of other co‐formulated drugs, such as pseudoephedrine. The application of the proposed method was extended to stability studies of LRT and DSL after exposure to different forced degradation conditions, such as acidic, alkaline and oxidative conditions, according to ICH guidelines. Copyright © 2011 John Wiley & Sons, Ltd.     

AcalPred: A Sequence-Based Tool for Discriminating between Acidic and Alkaline Enzymes

The structure and activity of enzymes are influenced by pH value of their surroundings. Although many enzymes work well in the pH range from 6 to 8, some specific enzymes have good efficiencies only in acidic (pH<5) or alkaline (pH>9) solution. Studies have demonstrated that the activities of enzymes correlate with their primary sequences. It is crucial to judge enzyme adaptation to acidic or alkaline environment from its amino acid sequence in molecular mechanism clarification and the design of high efficient enzymes. In this study, we developed a sequence-based method to discriminate acidic enzymes from alkaline enzymes. The analysis of variance was used to choose the optimized discriminating features derived from g-gap dipeptide compositions. And support vector machine was utilized to establish the prediction model. In the rigorous jackknife cross-validation, the overall accuracy of 96.7% was achieved. The method can correctly predict 96.3% acidic and 97.1% alkaline enzymes. Through the comparison between the proposed method and previous methods, it is demonstrated that the proposed method is more accurate. On the basis of this proposed method, we have built an online web-server called AcalPred which can be freely accessed from the website (http://lin.uestc.edu.cn/server/AcalPred). We believe that the AcalPred will become a powerful tool to study enzyme adaptation to acidic or alkaline environment.     

Isolation of the acidic and basic subunits of glycinin

The acidic and basic subunits of glycinin—the major storage protein of soybean seeds (Glycine max)—were isolated by a simple ion exchange chromatography method involving a two-step pH change of the elution buffer. Abnormal behaviour of the subunits in dodecyl sulfate polyacrylamide gel electrophoresis is suggested.     

Pulse-polarographic analysis of double-stranded RNA

The reducibility of double-stranded (ds) RNA of phage f2 and its thermally denatured form was studied by the derivative (differential) pulse polarography. It was shown that the denatured RNA produced at neutral and acidic pH values a peak at negative potentials. A peak of dsRNA was much smaller and appeared at more positive potentials. The polarographic behaviour of dsRNA basically agreed with that of DNA; the peaks of dsRNA and its denatured form were however better separated. The lowest amount of the denatured RNA necessay for the determination was about 50 ng. A good sensitivity of pulse polarography for changes in RNA conformation, including damages of the RNA double helix caused by initial attack of an enzyme, low doses of ionizing radiation, etc., was demonstrated. The method was used for testing dsRNA samples prepared for biological experiments; a correlation between the polarographic behaviour of the RNA samples and their antiviral and interferon-inducing activities was found.     

Interaction among proteins and peptide libraries in proteome analysis: pH involvement for a larger capture of species

When capturing proteins via combinatorial peptide ligand libraries, a method well known for drastically reducing the concentration of high-abundance proteins and substantially magnifying the signal of low-abundance species, thus leading to the discovery of a large number of proteins previously undetected in proteomes, we had constantly noticed that there would be a loss of species initially present in the untreated sample, to the tune of 5%, up to 15% in some cases. Such losses are a nuisance and hamper to some extent the unique performance of the method. In order to verify if such losses could be reduced and also to understand some mechanisms of the capture process, we introduce here an important variant to the capture operation, up to the present carried out in physiological saline at pH 7.2. In this novel protocol, the binding step is conducted at three different pH values, namely the standard one at pH 7.2, plus two additional processes, at acidic (pH 4.0) and alkaline (pH 9.3) pH values. Indeed the capture process is more extensive, with a number of additional species captured at the two pH extremes in sera and other proteomes. Interestingly, at pH 4.0 newly detected proteins were mostly acidic, while at the alkaline pH additional protein species were more evenly distributed throughout the pI range towards the alkaline area. The role of pH in the complex mechanism of binding among the hexapeptide library and the various proteomes being analyzed is discussed and evaluated. Due to significant changes in protein patterns with pH, recommendations are thus made to increase the possibility to find additional gene products illustrated by two examples (snake venom and leaf protein extract). Keeping under control the environmental pH when facing reproducibility studies or for comparative proteomics profiling is also a general rule suggested by this study.     

Probing the mechanism of amyloidogenesis through a tandem repeat of the PI3-SH3 domain suggests a generic model for protein aggregation and fibril formation

Aggregation of the SH3 domain of the PI3 kinase, both as a single domain and as a tandem repeat in which the C terminus of one domain is linked to the N terminus of another by a flexible linker of ten glycine/serine residues, has been studied under a range of conditions in order to investigate the mechanism of protein aggregation and amyloid formation. The tandem repeat was found to form amyloid fibrils much more readily than the single domain under the acidic conditions used here, and the fibrils themselves have higher morphological homogeneity. The folding-unfolding transition of the PI3-SH3 domain shows two-state behaviour and is pH dependent; at pH 3.6, which is near the pH mid-point for folding and only slightly below the isoelectric point of the protein, both the single domain and the tandem repeat spontaneously form broad distributions of soluble oligomers without requirement for nucleation. Under prolonged incubation under these conditions, the oligomers convert into thin, curly fibrils that interact with thioflavin-T, suggesting that they contain an organised beta-sheet structure. Under more acidic conditions (pH 2.0) where the proteins are fully denatured and carry a positive net charge, long, straight fibrils are formed in a process having a pronounced lag phase. The latter was found to be reduced dramatically by the addition of oligomers exceeding a critical size of approximately 20 molecules. The results suggest that the process of aggregation of these SH3 domains can take place by a variety of mechanisms, ranging from downhill formation of relatively amorphous species to nucleated formation of highly organised structures, the relative importance of which varies greatly with solution conditions. Comparison with the behaviour of other amyloidogenic systems suggests that the general mechanistic features outlined here are likely to be common to at least a wide variety of peptides and proteins.     

An Alcian blue (pH 2.5)-PAS-keratin immunoperoxidase method for the simultaneous demonstration of keratin and neutral and acidic mucosubstances

A method is proposed for the simultaneous staining of neutral and acidic, periodate reactive and nonperiodate reactive mucosubstances, glycogen and keratin in paraffin sections. Briefly, sections are stained by the Alcian blue (pH 2.5)-PAS method, followed by a peroxidase-antiperoxidase immunohistochemical stain for keratin. The proposed method modifies an existing method, and expands the range of polysaccharides and mucosubstances which may be demonstrated. The proposed method is easily performed within a single working day and promises to be of value in surgical pathology as well as in studies of bronchial carcinogenesis.     

An Alcian Blue (pH 2.5)-Pas-Keratin Immunoperoxidase Method for the Simultaneous Demonstration of Keratin and Neutral and Acidic Mucosubstances

A method is proposed for the simultaneous staining of neutral and acidic, periodate reactive and nonperiodate reactive mucosubstances, glycogen and keratin in paraffin sections. Briefly, sections are stained by the Alcian blue (pH 2.5)-PAS method, followed by a peroxidase-antiperoxidase imiminohistochemical stain for keratin. The proposed method modifies an existing method, and expands the range of polysaccharides and mucosubstances which may be demonstrated. The proposed method is easily performed within a single working day and promises to be of value in surgical pathology as well as in studies of bronchial carcinogenesis.     

Simple Process for the Reduction in the Nucleic Acid Content in Yeast

A simple one-step process for the nucleic acid reduction in Rhodotorula glutinis is described. The process consists of submitting the yeast cells to a heat treatment in an acidic (pH 2) spent medium. The optimal temperature for pH 2 medium is 90 C and the final nucleic acid content in treated yeasts was 1.2%. Heat treatment at acidic pH is preferred to that at alkaline pH because it offers a better protection for amino acids and crude protein, while being more efficient in lowering the nucleic acid level. The new process is economic and rapid and could be easily used for industrial application.     

Determination of the arginine content of proteins by the Sakaguchi procedure

The arginine contents of a variety of protein samples determined by the quantitative Sakaguchi method were in good agreement with the values obtained by quantitative ion-exchange chromatography, after acidic hydrolysis. One protein, α-chymotrypsin, while possessing a linear relationship between the colour obtained in the Sakaguchi test and the protein concentration, gave colour yields which corresponded to the apparent presence of an additional Sakaguchi-positive residue. The appearance of this phenomenon was found to be associated with the process of zymogen activation. Subsequently, it was found that two other serine esterases, trypsin and subtilisin, also exhibited this behaviour. Possible reasons for this have been discussed.     

Sodium-coupled sugar and amino acid transport in an acidic microenvironment

1. Nutrient transport mechanisms of lobster hepatopancreatic epithelial brush border membrane vesicles (BBMV) are strongly influenced by the acidic nature of the tubular lumen. 2. Sodium-dependent glucose uptake by BBMV was electrogenic and was stimulated at low pH by reducing sugar transport Ki, without affecting JM. 3. Glutamate was largely transported in zwitterionic form at pH 4.0 by an electrically silent cotransport mechanism with both Na and Cl. 4. Increased H+ concentration tripled the apparent membrane permeability to glutamate as well as the amino acid transport JM. 5. At pH 4.0 leucine was transported as a cation by two dissimilar carrier systems: a Na-independent process shared by polar amino acids, and an electroneutral Na-2Cl-dependent mechanism shared with non-polar amino acids. 6. A model is proposed for hepatopancreatic BBMV at acidic pH which employs ionic chemical gradients and membrane potential as nutrient transport driving forces.     

Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima

Although rapid auxin-induced growth of coleoptile sections can persist for at least 18 hours, acid-induced growth lasts for a much shorter period of time. Three theories have been proposed to explain this difference in persistence. To distinguish between these theories, the pH dependence for auxin-induced growth of oat (Avena sativa L.) coleoptiles has been determined early and late in the elongation process. Coleoptile sections from which the outer epidermis was removed to facilitate buffer entry were incubated, with or without 10 micromolar indoleacetic acid, in 20 millimolar buffers at pH 4.5 to 7.0 to maintain a fixed wall pH. During the first 1 to 2 hours after addition of auxin, elongation occurs by acid-induced extension (i.e. the pH optimum is <5 and the elongation varies inversely with the solution pH). Auxin causes no additional elongation because the buffers prevent further changes in wall pH. After 60 to 90 minutes, a second mechanism of auxin-induced growth, whose pH optimum is 5.5 to 6.0, predominates. It is proposed that rapid growth responses to changes in auxin concentration are mediated by auxin-induced changes in wall pH, whereas the prolonged, steady-state growth rate is controlled by a second, auxin-mediated process whose pH optimum is less acidic.     

Polarographic study of quinones formed during oxidation of caffeic and chlorogenic acids]

Polarographic behaviour of a number of substituted ortho-benzenquinones was studied in weakly acidic, neutral and weakly caustic solutions (up to pH --9.0). Half-wave potential depends on the solution pH, indicating protonization of the carbonyl groups of the quinones. Protonization is probably stipulated by the hydroxonium ions. Protonization may take place not only under the action of water, but also under the effect of other acidic components, as indicated by the dependence of E1/2 on the undissociated acetic acid centrifugation. The condensation and polymerization reactions are characteristic for the ortho-quinones. A shift of the quinone potential indicates dimerization of the electrode products.     

A mathematical model for the generation and control of a pH gradient in an immobilized enzyme system involving acid generation

An optimal pH control technique has been developed for multistep enzymatic synthesis reactions where the optimal pH differs by several units for each step. This technique separates an acidic environment from a basic environment by the hydrolysis of urea within a thin layer of immobilized urease. With this technique, a two-step enzymatic reaction can take place simultaneously, in proximity to each other, and at their respective optimal pH. Because a reaction system involving an acid generation represents a more challenging test of this pH control technique, a number of factors that affect the generation of such a pH gradient are considered in this study. The mathematical model proposed is based on several simplifying assumptions and represents a first attempt to provide an analysis of this complex problem. The results show that, by choosing appropriate parameters, the pH control technique still can generate the desired pH gradient even if there is an acid-generating reaction in the system.     

Modelling the effect of pH, acidulant and temperature on the growth rate of Yersinia enterocolitica

Growth of two pathogenic and one environmental serotype of Yersinia enterocolitica under acidic conditions and at 4 and 25 degrees C was investigated. At both temperatures the maximum growth inhibitory pH depended on the acidulant used and was in the order acetic greater than lactic greater than citric greater than sulphuric. At the lower temperature the maximum growth inhibitory pH was 0.3-0.5 pH units higher than at 25 degrees C. No difference was observed between the behaviour of pathogenic and environmental serotypes in this respect. Measurement of growth at a number of sub-optimal temperatures and pH values showed that the variation of growth rate with temperature could be represented by a square root plot. The effect of different pH values could be incorporated into the model by replacing the regression coefficient b by its relationship with pH. Values of maximum growth inhibitory pH derived from the model were in good agreement with experimental values with the exception of acetic acid.