Effect of stress exposure on the activation pattern of enkephalin-containing perikarya in the rat ventral medulla

We examined the effects of acute and chronic psychogenic stress on the activation pattern of enkephalin-containing perikarya in the rat ventrolateral medulla. Rats allocated to the chronic stress groups were subjected to 90 min of immobilization for 10 days. On the 11th day, the chronically stressed rats were exposed to homotypic (90-min immobilization) or to heterotypic but still psychogenic (90-min immobilization coupled to air jet stress) stress. The acute stress group was subjected once to an acute 90-min immobilization. For each group, the rats were anesthetized either before stress (time 0) or 90, 180, and 270 min after the onset of stress. Brain sections were then processed using immunocytochemistry (Fos protein) followed by radioactive in situ hybridization histochemistry (enkephalin mRNA). Following immobilization, the acute group displayed a marked increase in the number of activated enkephalin-containing perikarya within the paragigantocellularis and lateral reticular nuclei. This level of activation was sustained up to 180 min following the onset of the immobilization stress and had returned to baseline levels by 270 min from the initiation of the stress. However, this stress-induced activation of enkephalin-containing perikarya of the ventrolateral medulla was not seen following either homotypic or heterotypic stress in the chronically stressed group. These results provide evidence that enkephalin-containing perikarya of the ventrolateral medulla may constitute a potential circuit through which they regulate some aspect of the stress responses. Conversely, this enkephalinergic influence from the ventrolateral medulla was shown to be absent following chronic stress exposure. This would suggest a decrease in enkephalin inhibitory input originating from the ventrolateral medulla, thereby allowing a neuroendocrine and/or autonomic response to chronic stress.     

Protein immobilization on the surface of liposomes via carbodiimide activation in the presence of N-hydroxysulfosuccinimide

A method of the covalent immobilization of proteins on the surface of liposomes, containing 10% (by mol) of N-glutaryl phosphatidylethanolamine, is described. Carboxylic groups of liposomal N-glutaryl phosphatidylethanolamine were activated in the presence of water-soluble carbodiimide and N-hydroxysulfosuccinimide and reacted subsequently with protein amino groups. The liposome-protein conjugates formed contained up to 5 x 10(-4) mol protein/mol lipid. Lectins (RCA1 and WGA) upon immobilization on liposomes retained saccharide specificity and the ability to agglutinate red blood cells. The immobilization of mouse monoclonal IgG in a ratio of 3.5 x 10(-4) mol IgG/mol lipid was achieved. The liposome activation in the absence of N-hydroxysulfosuccinimide resulted in a 2-fold decrease of protein coupling yields.     

Elucidation of the effect of aptamer immobilization strategies on the interaction between cell and its aptamer using atomic force spectroscopy

The immobilization strategy of cell‐specific aptamers is of great importance for studying the interaction between a cell and its aptamer. However, because of the difficulty of studying living cell, there have not been any systematic reports about the effect of immobilization strategies on the binding ability of an immobilized aptamer to its target cell. Because atomic force spectroscopy (AFM) could not only be suitable for the investigation of living cell under physiological conditions but also obtains information reflecting the intrinsic properties of individuals, the effect of immobilization strategies on the interaction of aptamer/human hepatocarcinoma cell Bel‐7404 was successively evaluated using AFM here. Two different immobilization methods, including polyethylene glycol immobilization method and glutaraldehyde immobilization method were used, and the factors, such as aptamer orientation, oligodeoxythymidine spacers and dodecyl spacers, were investigated. Binding events measured by AFM showed that a similar unbinding force was obtained regardless of the change of the aptamer orientation, the immobilization method, and spacers, implying that the biophysical characteristics of the aptamer at the molecular level remain undisturbed. However, it showed that the immobilization orientation, immobilization method, and spacers could alter the binding probability of aptamer/Bel‐7404 cell. Presumably, these factors may affect the accessibility of the aptamer toward its target cell. These results may provide valuable information for aptamer sensor platforms including ultrasensitive biosensor design. Copyright © 2015 John Wiley & Sons, Ltd.     

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The characteristics of the UV-induced immobilization of oligonucleotides on nylon membranes and the efficiency of the enzymatic labeling of immobilized probes in heterophase identifying specific DNA sequences were studied. Oligonucleotides bound to short terminal oligothymidylates (up to 10 nt) through a flexible linker based on diethylene glycol phosphodiester are proposed as probes for immobilization on nylon. The presence of this fragment allows one to enhance the immobilization efficiency and reduce the UV-dependent degradation of the sequence-specific part of the probe by decreasing the irradiation dose needed for DNA immobilization. The optimal dose of UV irradiation is evaluated to be ∼0.4 J/cm² at 254 nm, which provides a high level of the hybridization signal for immobilized probes of various nucleotide sequences. It was found that nylon amide groups play a key role in the photoinduced fixation of oligonucleotides to the polymer surface, while its primary amino groups were not as responsible for the covalent binding of DNA as previously thought. Various additives in the membrane wetting solution were demonstrated to influence both the efficiency of the UV-induced immobilization and the functional integrity of immobilized probes. Other radical generating systems alternative to UV irradiation are shown to provide the immobilization of oligonucleotides on nylon membranes.     

Modulation of the distribution of small proteins within porous matrixes by smart-control of the immobilization rate

The distribution of enzymes attached to porous solid supports is a major concern in multienzymatic bioreactors. Herein, as proof of the concept that protein localization on porous surfaces can be controlled by tuning the protein immobilization rate. We study the distribution of two poly-histidine-tagged fluorescent proteins (His-GFP and His-mCherryFP) immobilized on different 4% crosslinked agarose-type carriers by confocal laser scanning microscopy. In this context, immobilization rate is easily modulated by controlling the (i) nature of physico-chemical interaction between protein and surface (reactive groups on surface), (ii) by controlling the reactive group density and (iii) by adding competitors to the immobilization process. His-GFP is 350-fold more rapid immobilized on agarose surfaces activated with either glyoxyl groups or chelates than the same matrix activated with primary amine groups instead. A similar effect is seen with agarose matrixes activated with lower glyoxyl densities that immobilize His-GFP roughly 350-fold slower than the corresponding highly activated matrix. When His-GFP is immobilized on agarose activated with chelates groups in presence of imidazol which competes with the protein for the reactive groups on the support, the immobilization rate is again 400-fold slower than when the same protein was immobilized on the same support but with no imidazol during the immobilization process. In all cases, it was observed that rapid immobilizations (quantitative immobilization in less than 10 min) located 100% of the loaded protein at the crown of the carrier beads, meaning that only the 10% of the bead radius was colonized by the protein. On the contrary, when immobilization is much slower, a homogeneous distribution is obtained, resulting in beads whose whole radius is occupied by the protein. Therefore, we set that the more rapid immobilization, the more heterogeneous distribution. All the knowledge gained in protein distribution by immobilization rate alteration of a single protein is applied to the co-immobilization of the two fluorescent proteins in order to develop four different co-immobilization patterns with an enormous applied potential to other multi-protein systems.     

Simultaneous purification and immobilization of Aspergillus niger xylanase on the reversibly soluble polymer Eudragit(TM) L-100

The non-covalent immobilization of a commercial preparation of xylanase from A. niger was carried out on a reversibly soluble-insoluble enteric polymer Eudragit(TM) L-100. The immobilization of the xylanase activity by adsorption was simultaneously accompanied by removal of cellulase activity since the latter did not bind to the polymer. Thus, the soluble enzyme derivative may be useful for treatment of paper pulp bleaching in paper industry. The immobilized xylanase retained 60% of its activity toward xylan as the substrate. No change was observed in the pH optimum (5.5) of the enzyme upon immobilization. Only marginal increase in the K(m) of the free enzyme (3.6 mg ml(-1) to 5.0 mg ml(-1)) upon immobilization on the soluble polymer reflected that the enzyme-substrate binding continues to be efficient in spite of the macromolecular nature of the substrate. Fluorescence spectroscopy and UV difference spectroscopy were used to probe the change(s) in the enzyme structure upon immobilization. This change in structure was correlated with the "effectiveness factor" of the enzyme activity. CD spectra also showed that the enzyme undergoes drastic changes in the structure.     

Enzyme immobilization on colloidal liquid aphrons (CLAs): the influence of system parameters on activity

The novel technique of immobilization of beta-galactosidase on colloidal liquid aphrons (CLAs) was investigated. CLAs are oil-in-water macroemulsions stabilised by a mixture of ionic and nonionic surfactants. Enzyme retention was found to be unaffected by changes in bulk phase pH and ionic strength, indicating that beta-galactosidase immobilization was due primarily to hydrophobic interactions. However, by varying the polarity of the internal solvent core, and the charge of the surfactants used in the formation of the CLAs, it was found that immobilization could be improved to almost 100% under certain conditions indicating that electrostatic interactions also affected immobilization to a lesser degree. Upon immobilization, it was found that there was a shift in the pH optimum of the enzyme, with the immobilized enzyme showing a broader range, and a maximal activity at higher pH. The immobilized beta-galactosidase displayed normal Michaelis-Menten dependence on substrate concentration, whilst also exhibiting superactivity for increased substrate concentrations. Activation energy was determined for the CLA immobilized enzyme, and it was found to decrease indicating that a conformational change had occurred that may account for the observed increase in activity. Finally, although the temperature profile of the immobilized enzyme was similar to the free enzyme, it was very stable, with a potential half-life of 3.6 years at 30 degrees C.     

Impact of spacers on the hybridization efficiency of mixed self-assembled DNA/alkanethiol films

The immobilization of DNA strands is an essential step in the development of any DNA biosensor. Self-assembled mixed DNA/alkanethiol films are often used for coupling DNA probes covalently to the sensor surface. Although this strategy is well accepted, the effect of introducing a spacer molecule to increase the distance between the specific DNA sequence and the surface has rarely been assessed. The major goal of this work was to evaluate a number of such spacers and to assess their impact on for example the sensitivity and the reproducibility. Besides the commonly used mercaptohexyl (C(6)) spacer, a longer mercapto-undecyl (C(11)) spacer was selected. The combination of both spacers with tri(ethylene)glycol (TEG) and hexa(ethylene)glycol (HEG) was studied as well. The effect of the different spacers on the immobilization degree as well as on the consecutive hybridization was studied using surface plasmon resonance (SPR). When using the longer C(11) spacer the mixed DNA/alkanethiol films were found to be more densely packed. Further hybridization studies have indicated that C(11) modified probes improve the sensitivity, the corresponding detection limit as well as the reproducibility. In addition two different immobilization pathways, i.e. flow vs. diffusion controlled, were compared with respect to the hybridization efficiency. These data suggest that a flow-assisted approach is beneficial for DNA immobilization and hybridization events. In conclusion, this work demonstrates the considerable impact of spacers on the biosensor performance but also shows the importance of a flow-assisted immobilization approach.     

Electric potential control of DNA immobilization on gold electrode

The assembly of synthetic, controllable molecules is one of the goals in nanotechnology. The primary objective of this contribution is to selectively immobilize DNA on gold via electric potential control. The self-assembly monolayer (SAM) was prepared with 2-aminoethanethiol (AET) on the gold electrode. A new approach based on electric potential was firstly used to control DNA immobilization covalently onto the SAM with the activation of 1-ethyl-3(3-dimethyl-aminopropyl)-carbodiimide (EDC) and N-hydroxysulfosuccinimide (NHS) in low ionic strength solution. The influence of electric potential on DNA immobilization was investigated by means of cyclic voltammogram, A.C. impedance, auger electron spectrometer as well as atomic force microscope (AFM) on template-stripped gold surface. The result proves that controlled potential can affect the course of DNA immobilization. More negative potential can restrain the DNA immobilization, while the more positive potential can accelerate the DNA immobilization. It is of great significance for the control of DNA self-assembly and will find wide application in the fields of DNA-based devices.     

Comparative studies on covalent and noncovalent immobilization of protein molecules on the surface of liposomes

Methods of enzyme molecules covalent immobilization on the surface of liposomes are suggested. The methods permit more protein molecules to be bound than traditional methods of noncovalent immobilization by means of adsorption or incorporation. The liposome membranes preserve their integrity during immobilization. At the same tame, the enzyme bound with the liposome surface via the “spacer” groups completely preserves its ability to interact with a specific macromolecular compound.     

Daily muscle vibration amelioration of neural impairments of the soleus muscle during 2 weeks of immobilization

V-wave, F wave and H-reflex responses of soleus were used to determine neural adaptations to 2-week immobilization and whether muscle vibration intervention during immobilization would attenuate the negative adaptations induced by immobilization. Thirty subjects were divided into the ankle immobilization group and the immobilization with muscle vibration group. Mechanical vibrations with constant low amplitude (0.3 mm) were applied (12 × 4 min daily) with a constant frequency of 100 Hz on the soleus muscle of the subjects in vibration group during the ankle immobilization period. Soleus maximal M-wave (Mmax) and H-reflex (Hmax) were evoked at rest. F-wave was recorded by supramaximal stimulation delivered at rest and V-wave during maximum voluntary contraction (MVC). The EMG during MVC was represented by its root-mean-square (RMS) value. Each subject was examined before and after 2 weeks of immobilization. Results showed that following 2 weeks of immobilization, Mmax, Hmax and F wave all did not change with immobilization in either group (P > 0.05). After 2 weeks of immobilization, significant reductions in V/Mmax (of 30.78%) (P < 0.01) and EMG RMS (24.82%) (P < 0.001) were found in the immobilization group. However, no significant changes occurred in the immobilization with muscle vibration group. Such findings suggested that 2 weeks of immobilization resulted in neural impairments as evidenced by the reduction in EMG and V wave, and that such decrease was prevented by the intervention of muscle vibration during the immobilization period.     

Preparation and properties of immobilized amyloglucosidase on nonporous PS/PNaSS microspheres

Nonporous polystyrene/poly(sodium styrene sulfonate) (PS/PNaSS) microspheres were used for immobilization of amyloglucosidase and the properties of immobilized enzyme was studied and compared with those of free enzyme. Sulfonated groups on the PS/PNaSS microspheres present a very simple, mild, and time-saving process for enzyme immobilization. Nonporous microspheres provide their surface for immobilization of enzyme and prevent the diffusion limitation problem in the pore. Despite the high concentration of bound enzyme the influence of immobilization on kinematic parameters, K(m) and V(max), is relatively low compare to other porous supports. Simple and time-saving immobilization procedure as well as the effects of pH and temperature on immobilized enzyme also showed that the PS/PNaSS microspheres could be good support.     

压电免疫传感器固定方法

抗原抗体在石英晶体电极表面固定而不丧失其活性是压电免疫传感器成功的关键,直接影响到它的灵敏度和可重复性等性质。介绍了压电免疫传感器传统的表面固定方法:主要有戊二醛交联法、自组装单分子膜法(SAM)、蛋白A固定法;以及国内外最新研究的不需通常固定化步骤的方法,主要是压电凝胶免疫分析法(LEPIA)和PEG压电凝胶分析法,并对这些方法的发展前景做了展望 。     

Improvement of the stability and selectivity of Rhizomucor miehei lipase immobilized on silica nanoparticles: Selective hydrolysis of fish oil using immobilized preparations

We report the effect of random and oriented immobilization of Rhizomucor miehei lipase (RML) on its functional properties. For this purpose, silica nanoparticles (MCM-41 and SBA-15) were prepared, characterized and functionalized by glycidyloxypropyl trimethoxysilane. Direct immobilization of RML on these supports was performed via the variety of amino acid residues on the surface of RML which promotes random immobilization. To perform oriented immobilization, partial modification of epoxy functionalized supports was carried out by introducing iminodiacetic acid groups followed by addition of Cu²⁺. In this way, immobilization is mainly directed via the most accessible histidine group, followed by intramolecular reaction of the other nucleophilic residues of the enzyme and the remaining epoxy groups on the support. The results showed higher thermal stability for immobilized derivatives compared to the soluble enzyme. Co-solvent stability of the derivatives was also studied in presence of six polar organic solvents (DMSO, THF, acetonitrile, 1-propanol, 2-propanol and dioxane). Influence of the immobilization procedure on activity and selectivity of the immobilized preparations was studied in selective hydrolysis of fish oil. All the derivatives discriminate between cis-5,8,11,14,17-eicosapentaenoic acid (EPA) and cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) in favor of EPA. Remarkable improvement in selectivity was obtained using oriented immobilization of RML.     

Effects of Immobilization Stress on the Background Impulse Activity of Locus Coeruleus Neurons

We studied in rats changes in the impulse background activity (BA) of locus coeruleus (LC) neurons after short- and long-term immobilization stress; distributions of LC neurons by the level of regularity of their BA, dynamics of spike trains, and pattern of histograms of interspike intervals (ISI) were taken into account. We also calculated the means of the main BA statistical indices. Both short- and long-lasting immobilizations resulted in drops in the mean frequency of background discharges of LC neurons to about half of the initial value. Two-hour-long immobilization evoked statistically significant shifts in the distribution of LC neurons by the level of regularity of their BA, while after longer (15 h) immobilization this distribution nearly returned to the initial pattern. Short-lasting immobilization exerted no significant effect on the dynamic characteristics of BA; statistically significant changes in this respect developed only after longer stress. After 15-h-long immobilization, we also observed a noticeable increase in the number of neurons with polymodal ISI distributions. Therefore, stress results in significant modifications of the temporal parameters of the BA of LC neurons; characteristics of the BA of these neurons should be considered neuronal correlates of the stress state.     

Distribution of immobilized enzymes on porous membranes

In this article, the results from a theoretical and experimental investigation of enzyme immobilization in porous membranes are reported. A theoretical model of the immobilization process, which accounts for restricted diffusion of enzyme in the pores of the membrane, has been developed. The model predicts the effect of immobilization kinetics and time of immobilization on the enzyme distribution in the pores of the membrane. The immobilization of glucose oxidase and glucose oxidase-biotin conjugate on porous alumina membranes was experimentally investigated. Enzyme uptake data was correlated to the theory to determine the rate constant of imobilization and the distribution of the enzyme in the pore. Immobilization studies were carried out for enzyme adsorption and for enzyme attachment by covalent coupling. The distribution of enzyme was experimentally studied by assembling five membranes in the diffusion cell. Following immobilization, the membranes were separated and each was assayed for activity. The amount of active enzyme present in each membrane yielded a discrete distribution that compared well with that predicted by theory. (c) 1992 John Wiley & Sons, Inc.     

Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(gamma-glutamic acid) by RSM

Poly(gamma-glutamic acid) (gamma-PGA) is a material of polymer. Immobilization of Candida rugosa lipase (Lipase AY-30) by covalent binding on gamma-PGA led to a markedly improved performance of the enzyme. Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were employed to evaluate the effects of immobilization parameters, such as immobilization time (2-6h), immobilization temperature (0-26 degrees C), and enzyme/support ratio (0.1-0.5, w/w). Based on the analysis of ridge max, the optimum immobilization conditions were as follows: immobilization time 2.3h, immobilization temperature 13.3 degrees C, and enzyme/support ratio 0.41 (w/w); the highest lipase activity obtained was 1196 U/mg-protein.     

Influence of cosolvents on the hydrophobic surface immobilization topography of Candida antarctica lipase B

The presence of cosolvents and co-solutes during the immobilization of lipases on hydrophobic supports may influence the extent of lipase immobilization and the long-term catalytic stability of the biocatalyst. Candida antarctica B lipase immobilization was examined on a hydrophobic surface, i.e., gold modified with a methyl-terminated, self-assembled alkylthiol layer. Lipase adsorption was monitored gravimetrically using a quartz crystal microbalance (QCM). Lipase activity was determined colorimetrically by following p-nitrophenol propionate hydrolysis. Adsorbed lipase topography was examined by atomic force microscopy (AFM). Lipase adsorption from low ionic strength aqueous buffer produced a uniform confluent protein monolayer. Inclusion of 10% (vol) ethanol in the buffer during immobilization resulted in a 33% adsorbed mass increase. Chemically similar cosolvents, all at 10% by volume in buffer, were also individually examined for their influence on CALB adsorption. Glycerol or 1-propanol increased mass adsorption by 10%, while 2-propanol increased mass adsorption by 33%. QCM dissipation values increased threefold with the inclusion of either ethanol or 2-propanol in the medium during lipase adsorption, indicating formation of multilayers of CALB. Partial multilayer formation using 10% ethanol was confirmed by AFM. Inclusion of 10% ethanol in the CALB immobilization buffer decreased the specific activity of the immobilized lipase by 37%. The formation of lipase multilayers in the presence of certain cosolvents thus results in lower specific activity, which might be due to either influences on lipase conformation or substrate active site accessibility.     

Improvement of the stability of alcohol dehydrogenase by covalent immobilization on glyoxyl-agarose

Immobilization of alcohol dehydrogenase (ADH) from Horse Liver inside porous supports promotes a dramatic stabilization of the enzyme against inactivation by air bubbles in stirred tank reactors. Moreover, immobilization of ADH on glyoxyl-agarose promotes additional stabilization against any distorting agent (pH, temperature, organic solvents, etc.). Stabilization is higher when using highly activated supports, they are able to immobilize both subunits of the enzyme. The best glyoxyl derivatives are much more stable than conventional ADH derivatives (e.g., immobilized on BrCN activated agarose). For example, glyoxyl immobilized ADH preserved full activity after incubation at pH 5.0 for 20h at room temperature and conventional derivatives (as well as the soluble enzyme) preserved less than 50% of activity after incubation under the same conditions. Moreover, glyoxyl derivatives are more than 10 times more stable than BrCN derivatives when incubated in 50% acetone at pH 7.0. Multipoint covalent immobilization, in addition to multisubunit immobilization, seems to play an important stabilizing role against distorting agents. In spite of these interesting stabilization factors, immobilization hardly promotes losses of catalytic activity (keeping values near to 90%). This immobilized preparation is able to keep good activity using dextran-NAD(+). In this way, ADH glyoxyl immobilized preparation seems to be suitable to be used as cofactor-recycling enzyme-system in interesting NAD(+)-mediated oxidation processes, catalyzed by other immobilized dehydrogenases in stirred tank reactors.     

Effect of chronic psychogenic stress exposure on enkephalin neuronal activity and expression in the rat hypothalamic paraventricular nucleus

This study tested the hypothesis that the activation pattern of enkephalinergic (ENKergic) neurons within the paraventricular nucleus of the hypothalamus (PVH) in response to psychogenic stress is identical whether in response to repeated exposure to the same stress (homotypic; immobilization) or to a novel stress (heterotypic; air jet puff). Rats were assigned to either acute or chronic immobilization stress paradigms (90 min/day for 1 or 10 days, respectively). The chronic group was then subjected to an additional 90-min session of either heterotypic or homotypic stress. A single 90-min stress session (immobilization or air jet) increased PVH-ENK heteronuclear (hn) RNA expression. In chronically stressed rats, exposure to an additional stress session (whether homotypic or heterotypic) continued to stimulate ENK hnRNA expression. Acute immobilization caused a marked increase in the numbers of Fos-immunoreactive and Fos-ENK double-labeled cells in the dorsal and ventral medial parvicellular, and lateral parvicellular subdivisions of the PVH. Chronic immobilization caused an attenuated Fos response ( approximately 66%) to subsequent immobilization. In contrast, chronic immobilization did not impair ENKergic neuron activation within the PVH following homotypic or heterotypic stress. These results indicate that within the PVH, chronic psychogenic stress markedly attenuates the Fos response, whereas ENKergic neurons resist habituation, principally within the ventral neuroendocrine portion of the nucleus. This suggests an increase in ENK effect during chronic stress exposure. Homotypic (immobilization) and heterotypic (air jet) psychogenic stressors produce similar responses, including Fos, ENK-Fos, and ENK hnRNA, within each subdivision of the PVH, suggesting similar processing for painless neurogenic stimuli.