Alteration in the brain electrical activity, diurnal sleep structure, and the rat behavior after immunization with bovine serum albumin conjugated with dopamine

Male Wistar rats (380-430 g) were immunized with bovine serum albumin conjugated with dopamine (2 mg/kg, 0.25 ml) mixed with Freund's adjuvant complete (0.25 ml) or with bovine serum albumin mixed with Freund's adjuvant complete in the same doses. One week after the immunization with bovine serum albumin conjugated with dopamine, irregular spike activity and high-amplitude spindles associated with the state of awake immobility were recorded in the rat neocortex and caudate putamen, the relative power of the electrical activity in the caudate putamen was decreased in the alpha band, while the relative power of the beta 1 in the cortical EEG was increased. In the structure of 4-hour diurnal sleep, a decrease in the mean duration of sleep episodes and a reduction in the REM sleep content were observed. The parameters of the electrical activity and diurnal sleep structure returned to normal during the following 4 weeks. The open-field behavior 2 weeks after the second immunization (without Freund's adjuvant complete) did not differ from that of the control rats immunized only with bovine serum albumin. Titres of antibodies to dopamine after the second antigen injection were 1:32-1:64 in the electrophysiological series and 1:128-1:256 in behavioral experiments.     

Effect of immunizing rats with a serotonin-bovine serum albumin conjugate on the structure of diurnal sleep and electrical activity of the brain]

Rats were immunized with bovine serum albumin conjugated with 5-hydroxytriptamine (5-HT). Two injections with a 2-week interval were carried out. At first, rats were injected subcutaneously (2 mg/kg, 0.25 ml) with Freund's adjuvant complete. The second injections (10 mg/kg intraperitoneally) was made 2 weeks later without the adjuvant. One week after the first antigen injection, there were a decrease in the relative power of the electrical activity of the caudate putamen, neocortex, and amygdala in the delta 2 and theta 2 bands and its increase in the alpha and beta-ranges. The number and duration (total and mean) of REM sleep episodes as well as REM sleep contribution in the total sleep duration decreased. These findings testify to a reduced functional activity of the central serotonergic system. Four-five weeks after the first injection, a tendency to a recovery of the bio-electrical spectral pattern and diurnal sleep structure was revealed. Five weeks after the first antigen injection, the titre of antibodies to 5-HT was 1:32-1:64.     

Alterations of spectral parameters of rat brain electrical activity after sciatic nerve section

Changes in electrophysiological brain characteristics accompanying the development of neurogenic pain syndrome induced by transsection of sciatic nerve were analyzed. At the maximum pain syndrome 3 weeks after the deafferentation, a reorganization of the brain electrical activity was observed in the limbic structures (hippocampus, amygdale, and nucleus accumbens), frontal cortex, and the caudate putamen. An increase in the relative spectral power of the delta and alpha bands and a decrease in the relative power of the beta2 band (as compared to baseline activity) took place. Alteration of the electrical activity in the limbic structures did not depend on manifestations of the neurogenic pain syndrome (autotomy). The increase in the relative spectral power of the alpha-band activity in all the structures under study suggests the involvement of the reticular thalamic nucleus in pathogenesis of neurogenic pain syndrome.     

Unfolding and refolding of bovine serum albumin induced by cetylpyridinium bromide

The interaction of bovine serum albumin (BSA) with cationic surfactant cetylpyridinium bromide (CPB) in aqueous solution (pH 7.00) was studied quantitatively with ultraviolet (UV)-visible, far-UV, and near-UV circular dichroism, fluorescence, small angle x-ray scattering, and nuclear magnetic resonance measurement. It was found that CPB at low and high concentrations could induce the unfolding and refolding of BSA, respectively. We suggest that in the unfolding process, there existed BSA-CPB complex with the "necklace and bead" structure in which the unfolded BSA wrapped around CPB micelles, and that the hydrophobic interaction between the complexes led to the formation of large aggregates. The aromatic headgroup of CPB interacted with the tryptophan residues of BSA, resulting in the aromatic ring stacking between BSA and CPB. During the refolding process, the BSA molecule was penetrated into the rod micelle of CPB and the hydrophobic moiety of the BSA molecule was exposed outside while its hydrophilic part was hidden inside, thereby disrupting the aromatic ring stacking.     

Interaction of ergosterol with bovine serum albumin and human serum albumin by spectroscopic analysis

This study was designed to examine the interactions of ergosterol with bovine serum albumin (BSA) and human serum albumin (HSA) under physiological conditions with the drug concentrations in the range of 2.99-105.88?μM and the concentration of proteins was fixed at 5.0?μM. The analysis of emission spectra quenching at different temperatures revealed that the quenching mechanism of HSA/BSA by ergosterol was the static quenching. The number of binding sites n and the binding constants K were obtained at various temperatures. The distance r between ergosterol and HSA/BSA was evaluated according to F?ster non-radioactive energy transfer theory. The results of synchronous fluorescence, 3D fluorescence, FT-IR, CD and UV-Vis absorption spectra showed that the conformations of HSA/BSA altered in the presence of ergosterol. The thermodynamic parameters, free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) for BSA-ergosterol and HSA-ergosterol systems were calculated by the van't Hoff equation and discussed. Besides, with the aid of three site markers (for example, phenylbutazone, ibuprofen and digitoxin), we have reported that ergosterol primarily binds to the tryptophan residues of BSA/HSA within site I (subdomain II A).     

Alterations of locomotor activity rhythm and sleep parameters in patients with advanced glaucoma

The aim of this study was to evaluate the effect of advanced glaucoma on locomotor activity rhythms and related sleep parameters. Nine normal subjects and nine age-matched patients with bilateral advanced primary open-angle glaucoma, >10 yrs since diagnosis, were included in this observational, prospective, case-control study. Patients were required to record the timing and duration of their sleep and daily activities, and wore an actigraph on the wrist of the nondominant arm for 20 d. Activity rhythm period, MESOR (24-h time-series mean), amplitude (one-half peak-to-trough variation), and acrophase (peak time), plus long sleep episodes during the wake state, sleep duration, efficiency, and latency, as well as mean activity score, wake minutes, and mean wake episodes during the sleep interval were assessed in controls and glaucomatous patients. Glaucomatous patients exhibited significant decrease in nighttime sleep efficiency, and significant increase in the mean activity score, wake minutes, and mean wake episode during the night. These results suggest that alterations of circadian physiology could be a risk to the quality of life of patients with glaucoma.     

Influence of air ions on brain activity induced by electrical stimulation in the rat

The brain induced activity was studied in 18 rats wearing chronically skull implanted electrodes. The stimulating factor was various electrical stimulations of the mesencephalic reticular activating formation, given during the slow wave state of sleep. The results of 300 stimulations were measured by amplitude and frequency changes in the EEG simultaneously recorded. Animals previously exposed to positive air ions (3 weeks 80,000 ions/ml) exhibited lowered excitability of the reticulocortical system. Significantly higher stimulations were necessary to induce arousal. Negative air ions induced more intricate effects: brain excitability was lowered when tested with weak stimulations, but normal when evaluated with medium high level stimilations. Sleep seems first more stable but as stimulation increases, arousal is soon as effective as in controls. These results are in agreement with others findings in behavioral fields and partly explains them.     

Method of determining proteolytic activity by using a conjugate of bovine serum albumin with peroxidase

The authors propose a method for determination of proteolytic activity, based on the hydrolysis of peroxidase-labeled molecules of bovine serum albumin immobilized on the surface of polystyrene microassay plates with the subsequent determination of peroxidase activity on the carrier or in the solution. The optimum conditions for the sorption of the labeled substrate have been established. The method permits the determination of bacillary alkaline protease at a concentration of 01. microgram/ml within 45 minutes. The determination of four proteases has demonstrated that this method shows good correlation with the routine one (r = 0.98), but is more sensitive and less time- and labor-consuming.     

Effects of oxidative modifications induced by the glycation of bovine serum albumin on its structure and on cultured adipose cells

Non-enzymatic glycosylation (glycation) and oxidative damages represent major research areas insofar as such modifications of proteins are frequently observed in numerous states of disease. Albumin undergoes structural and functional alterations, caused by increased glycosylation during non insulin-dependent diabetes mellitus, which is closely linked with the early occurrence of vascular complications. In this work, we first characterized structural modifications induced by the glycation of bovine serum albumin (BSA). A pathophysiological effect of glycated BSA was identified in primary cultures of human adipocytes as it induces an accumulation of oxidatively modified proteins in these cells. BSA was incubated in the presence or absence of physiological, pathological or supra-physiological concentrations of glucose at 37 degrees C for 7 weeks. Enhanced BSA glycation percentages were determined using boronate affinity columns. The occurrence of oxidative modifications was found to be enhanced in glycated BSA, after determination of the free thiol groups content, electrophoretic migration and infrared spectrometry spectra. An accumulation of carbonyl-modified proteins and an increased release of isoprostane were observed in cell media following the exposure of adipocytes to glycated albumin. These results provide a new possible mechanism for enhanced oxidative damages in diabetes.     

The adjuvant activity of lactoferrin in the generation of DTH to ovalbumin can be inhibited by bovine serum albumin bearing alpha-D-mannopyranosyl residues

Lactoferrin (LF) is an iron-binding glycoprotein present in the cytoplasmic granules of neutrophils and in external secretions of mammals. Although the biological role of human and bovine lactoferrin has been extensively studied, there is still uncertainty as to the nature and function of lactoferrin receptors. We recently determined that methyl-alpha-D-mannopyranoside given intraperitoneally (i.p.) could suppress the adjuvant activity of LF in the generation of delayed-type hypersensitivity (DTH) to ovalbumin (OVA). We concluded that the lactoferrin effects in DTH are mediated by carbohydrate-recognizing receptors like the mannose receptor (MR). This study indicates that subcutaneous (s.c.) administration of very small doses of the Man-bovine serum albumin (Man-BSA) complex, together with a sensitizing dose of the antigen, gives the same effects as i.p. administration of methyl-alpha-D-mannopyranoside. The latter is also a blocker of MR, although of a much lower affinity to the receptor than Man-BSA. The blocking of the adjuvant effect of LF by the Man-BSA complex (when given together with the sensitising dose of antigen) suggests that the function of antigen-presenting cells in the skin (presumably immature dendritic cells expressing MR) is inhibited. The results of our study indicate that a receptor with an affinity for mannose is essential for the mediation of adjuvant lactoferrin function in the generation of DTH.     

The convulsive activity of white rats after immunization with a conjugate of sidnofen and serum albumin

The influence of white rats immunization by a covalent conjugate of serum albumin with sydnophen on the seizure activity in the single and repeated injections of pentylenetetrazole was investigated. The immunization lowered the seizure activity in single injections of threshold doses, (60 mg/kg) of pentylenetetrazole. The repeated daily injections of the drug in subthreshold doses (30 mg/kg) inhibited the process of "kindling" effects formation.     

Characterization of bovine serum albumin glycated with glucose, galactose and lactose

The non-enzymatic reaction between reducing sugars and proteins, known as glycation, has received increased attention from nutritional and medical research. In addition, there is a large interest in obtaining glycoconjugates of pure well-characterized oligosaccharides for biological research. In this study, glycation of bovine serum albumin (BSA) by d-glucose, d-galactose and d-lactose under dry-heat at 60 degrees C for 30, 60, 120, 180 or 240 min was assessed and the glycated products studied in order to establish their biological recognition by lectins. BSA glycation was monitored using gel electrophoresis, determination of available amino groups and lectin binding assays. The BSA molecular mass increase and glycation sites were investigated by mass spectrometry and through digestion with trypsin and chymotrypsin. Depending on time and type of sugar, differences in BSA conjugation were achieved. Modified BSA revealed reduction of amino groups' availability and slower migration through SDS/PAGE. d-galactose was more reactive than d-glucose or d-lactose, leading to the coupling of 10, 3 and 1 sugar residues, respectively, after 120 minutes of reaction. BSA lysines (K) were the preferred modified amino acids; both K256 and K420 appeared the most available for conjugation. Only BSA-lactose showed biological recognition by specific lectins.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe³⁺-chelates, which initiated reactions that were dependent on membrane charge: Fe³⁺-EDTA and Fe³⁺-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe³⁺-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe³⁺-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Antioxidant effect of bovine serum albumin on membrane lipid peroxidation induced by iron chelate and superoxide

Albumin is supposed to be the major antioxidant circulating in blood. This study examined the prevention of membrane lipid peroxidation by bovine serum albumin (BSA). Lipid peroxidation was induced by the exposing of enzymatically generated superoxide radicals to egg yolk phosphatidylcholine liposomes incorporating lipids with different charges in the presence of chelated iron catalysts. We used three kinds of Fe3+-chelates, which initiated reactions that were dependent on membrane charge: Fe3+-EDTA and Fe3+-EGTA catalyzed peroxidation in positively and negatively charged liposomes, respectively, and Fe3+-NTA, a renal carcinogen, catalyzed the reaction in liposomes of either charge. Fe3+-chelates initiated more lipid peroxidation in liposomes with increased zeta potentials, followed by an increase of their availability for the initiation of the reaction at the membrane surface. BSA inhibits lipid peroxidation by preventing the interaction of iron chelate with membranes, followed by a decrease of its availability in a charge-dependent manner depending on the iron-chelate concentration: one is accompanied and the other is unaccompanied by a change in the membrane charge. The inhibitory effect of BSA in the former at high concentrations of iron chelate would be attributed to its electrostatic binding with oppositely charged membranes. The inhibitory effect in the latter at low concentrations of iron chelate would be caused by BSA binding with iron chelates and keeping them away from membrane surface where lipid peroxidation is initiated. Although these results warrant further in vivo investigation, it was concluded that BSA inhibits membrane lipid peroxidation by decreasing the availability of iron for the initiation of membrane lipid peroxidation, in addition to trapping active oxygens and free radicals.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.     

Endocytosis of native and glycosylated bovine serum albumin by duct cells of the rat parotid gland

Summary The ability of duct cells of the rat parotid gland to internalize bovine serum albumin (BSA) and several glycosylated albumins (glucosamide, galactosamide, fucosamide, lactosyl, p-aminophenyl-N-acetyl-D-glucosamide, p-aminophenyl-N-acetyl-D-mannopyranoside, p-aminophenyl-N-acetyl-D-galactosamide) was investigated. The various BSA preparations were infused into the gland via the main excretory duct, after which the tissues were fixed and prepared for light and electron microscopy. Immunolocalization of native BSA, as well as the glycosylated BSAs, was performed on thin sections, using an unlabeled antibody to BSA followed by protein A-colloidal gold. Gold particles were present over the lumina of both intercalated ducts and striated ducts, and over small endocytic structures and large vacuoles in the apical cytoplasm of both duct cell types. Endocytosis of the glycosylated BSAs by duct cells was greater than native BSA. Fucosylamide-BSA and mannopyranoside-BSA were taken up to a greater extent than the other glycosylated BSAs. Uptake by intercalated duct cells was greater than by striated duct cells, was independent of the concentration of the glycosylated BSA, and was reduced by an excess of the corresponding sugar. Striated duct cells showed some damage by the glycosylated BSAs that was concentration-dependent, and which was reduced in the presence of an excess of the corresponding sugar. These results suggest that endocytosis by salivary gland duct cells may involve specific recognition of carbohydrate residues and that the endocytosis of acinar secretory proteins observed in certain conditions may be due to increased and/or altered protein glycosylation.     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.