Partial purification and characterization of dihydrobenzophenanthridine oxidase from Eschscholtzia californica cell suspension cultures

The observation that upon elicitation cell suspension cultures of Eschscholtzia california showed a decrease of dihydromacarpine with a concomittant increase of macarpine led to the discovery of a novel enzyme which catalyzes the oxidation of dihydrobenzophenanthridines in the presence of oxygen. The enzyme was enriched approx. 70-fold. It has a pH-optimum of 7.0, an isoelectric point at pH 8.8, molecular weight of 56 kD and shows a high degree of substrate specificity. The enzyme obviously catalyzes the terminal step in the formation of benzophenanthridine alkaloids containing methylene dioxy substitutions in rings A and D.     

Changes in soluble carbohydrate level in Hordeum vulgare (L.) and Festuca pratensis (Huds.) calli treated with metabolites of Bipolaris sorokiniana (Sacc.) Shoem

Calli of spring barley (Hordeum vulgare L.) and meadow fescue (Festuca pratensis Huds.) were treated with metabolites of Bipolaris sorokiniana and then the level of soluble carbohydrates was estimated. Fructose and glucose occurred in the greatest amount in non-treated calli (control). Control tissue of both species responded to a change in culture conditions with fluctuation in the sugar level. Calli treated with fungal phytotoxins demonstrated rapid decrease in sugar content 1, 3 and 24 hrs after elicitation. Fescue calli, as less susceptible, showed moderate increase in carbohydrate level, yet it was still significantly lower than that in control. In barley very small amount of carbohydrates was observed as soon as 24 hrs after elicitation. In the elicited tissue of both species rapid increase in soluble carbohydrate level was noted in the 10^th hour. It is suggested that a defence response of barley and fescue takes place in two phases. The 1^st phase occurred between the 1^st and the 10^th hour after elicitation with phytotoxins and it seems to be an adaptation time to this stress factor. This stage is typical for both studied species. The 2^nd phase was observed after 10 hrs of pathogenesis. Its course may reflect a various sensitivity degree of both species to B. sorokiniana metabolites.     

A pseudo steady‐state model for galacto‐oligosaccharides synthesis with β‐galactosidase from Aspergillus oryzae

A pseudo steady‐state model for the kinetically controlled synthesis of galacto‐oligosaccharides (GOS) with Aspergillus oryzae β‐galactosidase is presented. The model accounts for the dynamics of lactose consumption and production of galactose, glucose, di, tri, tetra, and penta‐oligosaccharides during the synthesis, being able to describe the total GOS content in the reaction medium at the experimental conditions evaluated. Experimental results show that the formation of GOS containing only galactose residues is significant at high conversions of substrate, which was taken into account in the model. The formation of enzyme transition complexes was considered and reasonable assumptions were made to reduce the number of parameters to be determined. The model developed has 8 parameters; 2 of them were experimentally determined and the other 6 were estimated by fitting to the experimental data using multiresponse regression. Temperature effect on kinetic and affinity constants was determined in the range from 40 to 55°C, and the data were fitted to Arrhenius type equation. Parameters of the proposed model are independent from the enzyme load in the reaction medium and, differently from previously reported models, they have a clear biochemical meaning. The magnitude of the kinetic and affinity constants of the enzyme suggests that the liberation of galactose from the galactosyl–enzyme complex is a very slow reaction and such complex is driven into GOS formation. It also suggests that the affinity for sugars of the galactosyl–enzyme complex is higher than that of the free enzyme. Biotechnol. Bioeng. 2011;108: 2270–2279. © 2011 Wiley Periodicals, Inc.     

Experimental and theoretical investigations of submerged fermentation and synthesis of pectinolytic enzymes by Aspergillus niger

The growth of the microorganism and the production of the pectinolytic enzyme complex in a stirred 30-l biofermentor using the Aspergillus niger Rehbrücke strain were studied. The time courses of fermentation parameters (formation of biomass, consumption of carbon and inorganic nitrogen source, formation of pectinolytic enzymes) were measured. The formation of biomass showed a distinct lag phase, followed by a log phase with exponential growth and finally a stationary period when cell lysis was beginning. The uptake of the carbon source and inorganic nitrogen source by the A. niger cells corresponded to the time course of growth. The formation of pectinolytic enzymes took place in two steps. The first one was growth-bounded and finished with the end of the log phase of biomass growth. The second step of pectinolytic enzyme formation took place after the end of the catabolite repression of the carbon source and was not growth-bounded. On the basis of the experimental data a mathematical model of the fermentation process was developed. Comparison of the kinetics of the measured fermentation curves and the solution curves of the model showed qualitatively good agreement.     

Time course of induction of oxytocin messenger ribonucleic acid levels in the hypothalamic paraventricular nucleus of ovariectomized rats following gonadal steroid administration

The nonapeptide oxytocin (OT) is important for uterine contractility at parturition, milk ejection during lactation, and the induction of maternal behavior. OT messenger ribonucleic acid (mRNA) levels increase in the paraventricular and supraoptic nuclei (PVN and SON) of late pregnant and lactating rats and are modulated by the steroid milieu that accompanies these states. Specifically, sequential exposure to estradiol (E₂) and progesterone (P) followed by P withdrawal 48 hrs prior to sacrifice increases PVN, and to a lesser but significant degree, SON OT mRNA. To better define the time course of induction of OT mRNA levels following P withdrawal, ovariectomized Sprague-Dawley rats were treated with empty or steroid-filled capsules. On day 1, animals received an E₂-filled or empty capsule, followed by P-filled or empty capsules on day 3. On day 14, P-filled or empty capsules were removed and animals were sacrificed 24, 36, or 48 hrs later. The hypothalamic PVN were analyzed for OT mRNA by in situ hybridization histochemistry. Significant differences in PVN OT mRNA were found among the groups (P < 0.0001, Kruskal-Wallis). Animals in the 48 hr (P = 0.007) and 36 hr (P = 0.005), but not the 24 hr, steroid-treated groups had significantly increased OT mRNA relative to their respective sham-treated cohorts (Mann-Whitney U test). The relative abundance of PVN OT mRNA differed among the steroid-treated groups (Kruskal-Wallis, P < 0.0003), with highest levels at 48 hr. We conclude that increases in PVN OT mRNA occur by 36 hrs, and are highest at 48 hrs, after P withdrawal in the E₂-primed rat. Future studies will determine if OT-mediated changes in behavior or physiology that surround parturition are related to these changes in OT mRNA.     

Binding of porcine pancreatic secretory trypsin inhibitor to bovine beta-trypsin: a kinetic study

The kinetics of the formation of the complex between bovine β-trypsin and the porcine pancreatic secretory trypsin inhibitor (PSTI; Kazal-type inhibitor) was investigated following the spectral changes associated with the displacement of proflavine from the enzyme, upon inhibitor binding, between pH 3.5 and 8.0 (I = 0.1M) at 21 ± 0.5°C. With inhibitor in excess over the enzyme ([PSTI] ≥ 5 × [bovine β-trypsin]), the time course of the reaction corresponds to a pseudo-first-order process. Over the whole pH range explored, the concentration dependence of the rate is second order at low PSTI concentrations but tends to first order at high inhibitor concentrations. This behavior may be explained by a relatively fast pre-equilibrium followed by a limiting first-order process. Values of kinetic parameters for PSTI binding to bovine β-trypsin depend, between pH 3.5 and 8.0, on the acid–base equilibrium of a single ionizing group (probably His-57 of bovine β-trypsin) that undergoes an acidic pK_a shift from 7.0 in the free bovine β-trypsin to 5.5 in the enzyme:PSTI complex. Kinetics of the bovine β-trypsin:PSTI adduct formation has been analyzed and compared with that of other (pro)enzyme:inhibitor reactions. Considering the known molecular structures of free serine (pro)enzymes, of Kazal- and Kunitz-type inhibitors, as well as of their complexes, the binding behavior of PSTI to bovine β-trypsin has been related to the inferred stereochemistry of the proteinase:inhibitor contact region.     

Elicitation of benzophenanthridine alkaloid synthesis in Eschscholtzia cell cultures

Quaternary benzophenanthridine alkaloids (sanguinarine, chelerythrine, chelirubine, chelilutine and macarpine) are specifically induced by cell wall components of Penicillium and Saccharomyces in a colorless strain of Eschscholtzia californica cell suspension cultures. Classical elicitors such as the Phytophthora megasperma elicitor are inactive. The alkaloid synthesis is, however, strongly induced by certain polypeptide antibiotics. Out of 190 tested plant species the yeast elicitor provoked benzophenanthridine synthesis in 13 cultures. One of the branch point enzymes, namely the berberine bridge enzyme, catalysing the formation of (S)-scoulerine from (S)-reticuline, is strongly stimulated during the elicitation process. These results clearly demonstrate the induction of the benzophenanthridine biosynthetic pathway by microbial elicitors.Abbreviations ACC 1-Aminocyclopropane-1-carbonic acid - EDTA Ethylenediaminetetraacetic acid - LS-medium Linsmaier and Skoog medium - Pmg Phytophthora megasperma     

A Model for the Mechanism of Enzyme Induction

A sequence of reactions is postulated from which are derived equations describing the time course of enzyme induction. The model also yields the observed effect of the inducer concentration on the time constant and final rate of enzyme synthesis. Features of the model are: (a) The inducer acts first to release the protein forming template from its site of synthesis on the gene. (b) The inducer is involved again in the equilibrium dissociation of the free template-inducer complex which is utilized in the synthesis of the enzyme-forming unit. (c) The final enzyme-forming unit is unstable and must be synthesized continuously to maintain enzyme synthesis.     

A Brownian model of glutamate diffusion in excitatory synapses of hippocampus

We simulated the diffusion of glutamate, following the release of a single vesicle from a pre-synaptic terminal, in the synaptic cleft by using a Brownian diffusion model based on Langevin equations. The synaptic concentration time course and the time course of quantal excitatory post-synaptic current have been analyzed. The results showed that they depend on the number of receptors located at post-synaptic membrane. Their time course are dependent both on the total number of the post-synaptic receptors and on the eccentricity of the pre-synaptic glutamate vesicle.     

Localization of aldolase activity in the embryos of loach]

The activity of aldolase was determined in different parts of the loach (Misgurnus fossilis L.) embryo at the stages from the formation of axial organs till the beginning of embryonic movements (from 19 till 38 hrs of development at 21.5 degrees). In all parts of the embryo, the activity of aldolase at first decreased (21-23 hrs) and then increased. The region of somites is characterized by the highest absolute and specific activity at all developmental stages. The increase in the number of somites is accompanied by the fall and subsequent rise of aldolase activity. In the somites of different degree of differentiation, the enzyme activity changes in a similar way. Hence, there is no correlation between the morphological and biochemical differentiation of somites. Differences in the specific aldolase activity between the anterior and posterior halves of the embryo and the regions of head, somites and tail were found at the stages of 19-23 hrs of development. The maternal aldolase only is present at these stages, as was shown earlier. It means that the early stages of biochemical differentiation may be realized not by means of differential activation of genes controlling the enzyme, but by means of regylation of translation on the templates stored in oogenesis.     

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.     

Competitive and uncompetitive inhibitors simultaneously acting on an autocatalytic zymogen activation reaction

The time course of the residual enzyme activity of a general model consisting of an autocatalytic zymogen activation process inhibited by an irreversible competitive inhibitor and an irreversible uncompetitive inhibitor has been studied. Approached analytical expressions which furnish the time course of the residual enzyme activity from the onset of the reaction depending on the rate constants and initial concentration have been obtained. The goodness and limitations of the analytical equations were checked by comparing with the results obtained from the numerical integration, i.e. with the simulated progress curves. A dimensionless parameter giving the relative contributions of both the activation and the inhibitions routes is suggested, so that the value of this parameter determines whether the activation or the inhibitions routes prevail or if both processes are balanced during the time for which the analytical expressions are valid. The effects of the initial zymogen, free enzyme and inhibitors concentrations are analysed. Finally an experimental design and kinetic data analysis is proposed to evaluate simultaneously the kinetic parameters involved and to discriminate between different zymogen activation processes which can be considered particular cases of the general model.     

Chemical kinetics of induced gene expression: activation of transcription by noncooperative binding of multiple regulatory molecules

A chemical kinetics model is described for the regulation of gene expression by the progressive binding of regulatory molecules to specific binding sites on DNA. Chemical rate equations are formulated and solved for the accumulation of regulatory molecules on DNA, the change in the level of induced mRNA, and the change in the level of the encoded protein in the activated tissue. Some special cases are examined, including that of an activation threshold created by a requirement for the binding of a minimum number of regulatory molecules prior to gene activation. Experimental data for several hormone-activated genetic systems are analyzed in the frame of the proposed model, and kinetic parameters are predicted. The model accounts for a number of experimental characteristics of hormone-inducible genetic systems, including the existence of a lag in the time course of mRNA accumulation, the sigmoidal curve of induced mRNA kinetics, the effect of hormone on mRNA stabilization, and the induction parameters observed when hormone analogues are used. The model also provides an explanation for the phenotypes of genetic variants with altered inducibility as changes in the molecular kinetic parameters of gene activity.     

TheN haplotype of the murineβ-glucuronidase gene is altered in both its systemic regulation and its response to androgen induction

A new haplotype of the -glucuronidase gene complex, [Gus]^N, has been characterized following its transfer from the PAC/Cr strain to the standard strain C57BL/6J. TheN haplotype contains a novel structural gene allele which encodes an allozyme differing from all previously characterized allozymes in both size and charge. Altered systemic regulation is exhibited by the [Gus]^N haplotype. Multiple tissues contain levels of GUS protein that are 60±15% those found in the standardB haplotype. The regulatory mechanism for reduction is complex, involving tissue-specific changes in both enzyme synthesis and enzyme turnover. The changes in GUS protein synthesis do not result from changes in GUS mRNA levels. Instead, the amount of mature enzyme formed per mRNA molecule, or translational yield, is altered. These regulatory changes parallel those seen in other systemic regulatory variants of GUS which are also altered in translational yield. A commonality of mechanism among systemic regulatory variants of this gene is suggested. TheN haplotype is also exceptional in the nature of its response to androgenic induction in kidney proximal tubule epithelial cells. The time course for GUS induction consists of a lag period followed by a progressive increase in mRNA, rate of enzyme synthesis, and enzyme activity. For the [Gus]^N haplotype the lag is of an exceptionally short duration and the plateau is of a greater magnitude than for any haplotype previously described.This work was supported by United States Health Service Research Grant GM 31656.     

The kinetics of invertase action

A method of obtaining better fits to experimental curves for the hydrolysis of sucrose in the presence of invertase is found by assuming that the reaction is first-order, but that there is an initial time delay before this form is assumed. More rigorously, a system of two differential equations describing the reaction is established, assuming that the formation of enzyme-substrate complex is irreversible, and a solution is obtained relating, at any time, the concentration of the products, the total concentration of complex which has been formed in that time, and the time. This solution is compared with other equations for the reaction and is used to calculate reaction constants. Curves showing the concentration of complex existing at timet are given and are employed in calculating an “equivalent” enzyme concentration. This expression is discussed. Supported in part by a contract from the Atomic Energy Commission.     

Sequential changes in ornithine decarboxylase thymidine kinase, and other enzyme activities in regenerating liver in rats on controlled feeding schedules.

The changes in activity of five enzymes including ornithine decarboxylase (ODC), tyrosine aminotransferase (TAT), thymidine kinase (TK), ornithine aminotransferase (OAT) and serine dehydratase (SDH) in the early stage of the regenerating rat liver have been studied under a controlled feeding and lighting schedule. The first three enzyme activities were stimulated sequentially by partial hepatectomy. The earliest response was observed in ODC activity. A significant increase in this enzyme activity was observed at 2 hrs and the maximal level was at 4 hrs after the operation. TAT began to increase at 4 hrs and the maximal level was at 8 hrs. The TK activity was induced at about 24 hrs and the highest value was at 48 hrs after partial hepatectomy.A significant decrease in OAT activity was observed at 24 hrs after the operation and subsequently. Although a decrease in SDH activity was also observed this decrease did not seem to correlate directly with the regeneration process, since a lowered level of the enzyme activity was also found in the sham operated group.     

Visualization of elicitor-binding loci at the plant cell surface

We describe a method which allows the visualization of elicitor-binding loci at the surface of plant protoplasts. Prerequisites for this method are the preparation of protoplasts under conditions of minimal proteolysis, and the availability of antibodies against either the elicitor itself or against the fluorescein portion of elicitor-fluorescein conjugates. Silver enhancement is used to amplify the visibility of 5-nm gold particles which are attached to an appropriate secondary antibody. Bound elicitor can then be visualized by epipolarization microscopy. This method, designated SEIG-EPOM (for silver enhanced immunogold as viewed by epipolarization microscopy), has been applied to protoplasts of parsley (Petroselineum cirspum L.), using the Phytophthora megasperma elicitor, and soybean (Glycine max Merr.) using polygalacturonic acid elicitor isolated from citrus pectin. We have been able to estimate the number of specific binding loci as being less than 100 per protoplast. Such loci possibly represent clusters of individual elicitor-receptor complexes. Structurally related elicitors have been shown to compete effectively for binding sites. The latter are sensitive to proteolysis, as is the elicitation response of protoplasts.Dedicated to Professor Peter Sitte on the occasion of his 65th birthday     

The Lipid Peroxidation Product 4-Hydroxynonenal is Formed by - and is able to Attract - Rat Neutrophils in vivo

4-Hydroxynonenal (HNE), a major aidchydic product of lipid peroxidation, is a chemoattractant for neutrophilic polymorphonuclear granulocytes in vitro. The question was studied, whether HNE is formed during the ingress of neutrophils in the Sephadex model of inflammation. The polydextrane Sephadex G-200, which causes an acute aseptic traumatic inflammation, was injected subcutaneously into rats. The implants were excised 6-36 hours later, and the neutrophils separated from the exsudate by centrifugation. After extraction with dichloromethane HNE was identified in the exsudate by non-derivative reversed phase HPLC in combination with on-line uv-spectroscopy. The concentration of HNE in the inflammatory focus did not correlate with the number of neutrophils present. While the peak of HNE coincided with the time point of the highest turnover rate of neutrophils (0.13 μM at 6 hrs after implantation), the highest number of neutrophils (about 100 million cells) occurred not earlier than 18 hrs later (24 hrs after onset of inflammation).

When neutrophils were isolated from the inflammatory focus and stimulated with Zymosan, they were able to produce HNE in vitro depending on the time of isolation. The highest production of HNE (0.17 μM) by phagocyting neutrophils was observed at the shortest inflammation time studied (3 hrs). In order to compare these results with the oxidative burst of neutrophils the formation of superoxide was also measured by the cytochrome c reduction assay in vitro. The maximum of the production rate of superoxide anion was observed at the same inflammation time (6 hrs), when the HNE maximum occurred. Cells which ingressed earliest (at 3 hrs) showed the highest production rate of superoxide per cell (307 × 10^-18 moles per cell and 30min).

The ability of HNE to attract neutrophils in vivo was studied by adding synthetic HNE to the Sephadex gel and measuring the ingression of neutrophils afterwards. The application of 1 μM HNE in the focus did not change the number of neutrophils but 10 μM HNE increased the cell number by a factor of 3.

The results indicate that HNE is not only a chemoattractant for rat neutrophils in vitro but also in vivo. It is suggested that HNE is produced by selfdestruction of neutrophils during a traumatic inflammation and its production seems to be tightly connected to the oxidative burst of neutrophils. The idea of HNE as part of an autocatalytic cycle is supported whereby neutrophils which immigrate into an inflammatory focus produce HNE which stimulates the ingress of new neutrophils.     

Validation of computational approach to study monomer selectivity toward the template Gallic acid for rational molecularly imprinted polymer design

Gallic acid (GA) is important for pharmaceutical industries as an antioxidant. It also finds use in tanning, ink dyes and manufacturing of paper. Molecularly imprinted polymers (MIP), which are tailor made materials, can play an excellent role in separation of GA from complex matrices. Molecular recognition being the most important property of MIP, the present work proposes a methodology based on density functional theory (DFT) calculations for selection of suitable functional monomer for a rational design of MIP with a high binding capacity for GA. A virtual library of 18 functional monomers was created and screened for the template GA. The prepolymerization template-monomer complexes were optimized at B3LYP/6-31G(d) model chemistry and the changes in the Gibbs free energy (ΔG) due to complex formation were determined on the optimized structures. The monomer with the highest Gibbs free energy gain forms most stable complex with the template resulting in formation of more selective binding sites in the polymeric matrix in MIPs. This can lead to high binding capacity of MIP for GA. Amongst the 18 monomers, acrylic acid (AA) and acrylamide (AAm) gave the highest value of ΔG due to complex formation with GA. 4-vinyl pyridine (4-Vp) had intermediate value of ΔG while, methyl methacrylate (MMA) gave least value of ΔG due to complex formation with GA. Based on this study, the MIPs were synthesized and rebinding performance was evaluated using Langmuir-Freundlich model. The imprinting factor for AA and AAm based MIPs were 5.28 and 4.80 respectively, 4-Vp based MIP had imprinting factor of 2.59 while MMA based MIP exhibited an imprinting factor of 1.95. The experimental results were in good agreement with the computational predictions. The experimental data validated the DFT based computational approach.