Characterisation and pharmacological sensitivity of antigen arthritis induced by methylated bovine serum albumin in the rat.

The optimum conditions for the induction of antigen-induced arthritis in the rat have been studied. Two immunisations with methylated bovine serum albumin (mBSA) (0.5 mg) in Freund's complete adjuvant (FCA) containing 0.375 mg Mycobacterium tuberculosis followed by an intra-articular injection of mBSA (0.5 mg) led to the production of a chronic, erosive arthritis. The development of the arthritis was associated with the appearance of T lymphocytes and other inflammatory cells in the synovium. Male and female animals were equally susceptible to the disease. Prednisolone, indomethacin and methotrexate inhibited the development of the arthritis but ibuprofen and D-penicillamine were without any significant effect.     

阳离子化牛血清白蛋白致大鼠慢性肾炎模型的研究

目的 探索利用阳离子化牛血清白蛋白(C-BSA)复制膜性肾病模型的可行性,探讨模型特点,以及模型对各项指标的影响及规律.方法 模型组隔日尾iv给予C-BSA溶液5 mg/只,持续6周;造模停止后继续观察4周.造模期第4、6周以及观察期第8、10周采集血样、尿样,测定血清生化指标、血常规指标、尿蛋白指标等,并留取相关脏器,计算脏器系数,并进行病理学检查.结果 造模期时,血清总蛋白、白蛋白明显下降,血脂明显升高,肌酐、尿素氮变化不明显;血浆黏度明显升高,全血黏度变化不大;尿蛋白明显增加,尿量减少,尿白细胞增加;血红细胞、血红蛋白降低;肾系数、脾系数明显提高,体质量明显下降;模型组动物肾脏可见不同程度的肾小管蛋白管型、肾小管扩张、再生肾小管、间质炎细胞浸润、肾小球鲍曼氏囊增厚.造模结束进入观察期后,模型出现一定程度的恢复,各项指标均向正常值趋近.结论 成功复制了大鼠C-BSA模型,模型稳定,临床表现典型,可重复性高.     

Determination of loperamide in rat plasma and bovine serum albumin by LC

A rapid, isocratic liquid chromatographic (LC) method was developed for the determination of loperamide (Lop) in solutions of bovine serum albumin (BSA) and rat plasma. Prior to LC analysis, BSA solutions or rat plasma samples were treated with metaphosphoric acid to precipitate protein. Supernatant was directly injected onto a C18 reverse phase column and loperamide was monitored by a UV detector set at 195 nm. The concentrations of Lop in both rat plasma and BSA solution samples were determined by comparison with their calibration curves, which were generated from the peak area ratio of Lop to internal standard, clomipramine versus loperamide concentration. The calibration curves were linear in the range 0-3.0 microg ml(-1) of Lop for the BSA solution sample and 0-1.0 microg ml(-1)for the rat plasma sample. Overall recoveries of loperamide added to BSA and rat plasma samples were 101.4 and 95.5%, respectively. The method is simple (no extraction), rapid (22 min separation time), sensitive (the detection limit of loperamide is 50 ng ml(-1) for the BSA solution sample and 100 ng ml(-1) for the rat plasma sample), reproducible (within-day R.S.D. of 2.59-7.11%, among-day R.S.D. of 1.25-5.97%), and suitable for routine analysis of loperamide in rat plasma and BSA solution samples.     

阿糖胞苷引起毛囊损伤离体模型的建立以及他克莫司对其的逆转作用

目的建立阿糖胞苷(Ara-c)诱导毛囊损伤的离体器官培养模型,并观察他克莫司(FK506)对Ara-c诱导的小鼠触须毛囊损伤的逆转作用.方法用离体器官培养的方法在倒置显微镜底下每日测量空白组、FK506 Ara-c和单纯Ara-c作用下毛囊生长长度、记录生长天数,以及液相闪烁仪测量同位素^3H-TdR的掺入率.结果 Ara-c10mg/L和25mg/L的浓度,作用2.5h能明显抑制毛囊生长和DNA合成,缩短毛囊在体外的生长时间,抑制毛囊球部细胞增殖.0.01～0.3mg/L的FK506能改善10mg/L Ara-c引起的毛囊生长和DNA合成的抑制,体外生长时间缩短以及毛囊球部细胞增殖抑制.0.1mg/L的FK506对25mg/L Ara-c引起的毛囊损伤也有相似的改善作用.结论 Ara-c诱导毛囊损伤的离体模型可以用于研究化疗脱发的机制和某些因子及药物对它的干预作用,FK506在体外对Ara-c诱导的毛囊损伤有修复作用,是治疗化疗后脱发的潜在药物.     

The effect of benzopyrone on the bovine albumin induced exudative peritonitis in the rat (author's transl)

The antiphlogistic effect of the benzopyrone drug "Venalot" could be proven in the model of the exsudative immune-peritonitis in the rat, caused by bovine serum-albumin. Under the action of the drug the exudate and the amount of proteins in the peritoneal fluid were reduced; an important reduction of the adhesions occurred, too. The differences between the mean values and observations in the control and in the treated group were statistically significant (P less than 0.001).     

FK506 ameliorates spontaneous locomotor activity in collagen-induced arthritis: implication of distinct effect from suppression of inflammation

FK506 (tacrolimus), an immunosuppressive drug, improves quality of life (QOL) for patients with rheumatoid arthritis (RA). However, the mechanism of FK506 behind the improvement in QOL is still uncharacterized. To explain the improvement of QOL by FK506, we investigated the effect of FK506 on spontaneous locomotor activity in rats with collagen-induced arthritis (CIA). CIA was induced in 7- to 8-week-old female Lewis rats by immunization with bovine type II collagen. After initiation of paw inflammation (paw swelling, histopathological analysis), CIA rats were therapeutically administered FK506 or methotrexate (MTX) from day 15. Therapeutic treatment with FK506 ameliorated spontaneous locomotor activity without suppressing paw inflammation in CIA rats from day 27. FK506 also improved hyperalgesia and grip strength from day 27. Therapeutic treatment with MTX did not improve spontaneous locomotor activity, and simultaneously did not recover hyperalgesia or grip strength in CIA rats. Our results indicate that spontaneous locomotor activity in CIA rats correlates mainly with hyperalgesia and muscle strength, but not paw inflammation, implying that therapeutic treatment with FK506 ameliorates spontaneous locomotor activity via improvement of hyperalgesia and muscle strength in CIA rats.     

FG-FK506药物缓释系统对大鼠坐骨神经损伤修复作用的实验研究

目的探讨在大鼠坐骨神经损伤后,局部应用纤维蛋白凝胶（FG）-他莫克司（FK506）药物缓释系统对神经再生的影响,为FK506的临床应用提供一种可行的给药方式。方法取24只成年SD大鼠随机分为4组,切断左侧坐骨神经后随即进行显微吻合,其中实验组（B组）在吻合口周围应用FG-FK506药物缓释系统,另设3组对照（A、C、D组）,分别作为空白对照组及全身应用FK506组、局部单纯应用FG组,术后观察各组大鼠的大体形态、步态、关节活动情况。术后12周测量坐骨神经诱发动作电位幅度和传导速度,计算小腿肌肉湿重恢复率。结果术后各组大鼠均全部成活,术侧肢体活动受限,术侧负重区出现不同程度红肿及溃疡,肌肉萎缩,实验组大鼠肢体功能及肌肉萎缩恢复最快。术后12周实验组坐骨神经与周围组织无粘连,对照组与周围组织轻度粘连,远端神经略细。大鼠坐骨神经的复合肌肉动作电位波幅、运动神经传导速度及小腿三头肌湿重恢复率均高于对照组。结论 FG-FK506药物缓释系统在大鼠坐骨神经再生中起到明显促进作用,未引起全身及局部明显免疫抑制作用,是一种有效的给药方式。     

The effect of hydrotropic agents on the heat coagulation of bovine serum albumin

The effect of two hydrotrophic solubilizers on the heat coagulation of bovine serum albumin (BSA) has been investigated. Photon correlation spectroscopy indicated possible unfolding of BSA molecules in solutions of sodium benzoate and sodium salicylate at 25 degrees C. The effect of these hydrotropes on the heat coagulation of BSA was concentration-dependent. Relatively low concentrations stabilized the protein structure as indicated by the increase in the transition temperature(Tm) and induced gelation at temperatures and BSA concentrations lower than those required in the absence of hydrotropes. Higher concentrations of the hydrotropes considerably reduced Tm and inhibited gelation of BSA, the effect of sodium salicylate being more pronounced, as was the lower aggregation rate of BSA. The behaviour of these hydrotropes as protein denaturants differs from that of neutral electrolytes but is similar to that of concentrated solutions of urea.     

Adsorption of cationized bovine serum albumin onto epithelial crypt fractions of the rat colon.

The purpose of the study was to characterize mucosal attachment of a cationized model protein, bovine serum albumin (BSA), onto the various fractions of colonic crypts epithelium in the rat. BSA was labeled with fluorescein isothiocyanate (FITC) and its surface net electric charge was modified from negative to positive. Attachment of the cationized protein (CF-BSA) onto rat colonic epithelium was performed by incubation of colonic everted sacs in medium containing cationized or non-cationized FITC-labeled BSA. Using a nonenzymatic isolation procedure, colonocytes were harvested from five horizontal fractions of the colonic crypts. BSA adhesion to the isolated colonocytes was quantified spectrofluorometrically. In addition, the effect of increasing concentrations of Mg(2+) on the adsorption of the cationized BSA onto the surface of colonic epithelium was evaluated by measuring its ability to displace the adhered BSA from its binding sites. BSA cationization facilitated protein adherence to the colon epithelium in a crypt depth-dependent manner. The largest extent of adherence was observed in the outer layer (first fraction) of the colon. Binding persisted to approximately half the depth of the crypts. The relation between CF-BSA concentration in the incubation medium and the amount of CF-BSA adsorbed onto the colonic epithelium was exponential in nature. The addition of electrolyte (Mg(2+)) caused a detachment of the CF-BSA. The adsorption process was characterized by Langmuir's adsorption isotherm. It is concluded that cationized BSA could be useful as a targetable drug platform in cases where the target site is the gastrointestinal epithelium.     

Effects of bovine serum albumin and recirculation rate on the uptake of propranolol by rat perfused lung

Lungs isolated from 7-week-old rats were perfused with pH 7.4, oxygenated Krebs-Ringer bicarbonate buffer solution containing 2.5 micrograms mL-1 of propranolol and 3 to 5% BSA at the recirculation rate of 4 to 16 mL min-1, at 37 degrees C for 60 min. The extent of propranolol metabolism after 60 min was less than 2.3% of the initial load under any in-vitro perfusion condition. Therefore, the amount disappearing from the perfusion medium was considered as being predominantly that taken up by tissue. Under all experimental conditions, perfusate drug level declined bi-exponentially with time. Apparent in-vitro pulmonary clearance of propranolol was not affected by the increase of BSA level from 3 to 5%. When the perfusate BSA level was fixed at 3%, the lowest recirculation rate (4 mL min-1) yielded the smallest clearance (about 0.15 mL min-1 g-1) but almost constant clearance value (about 0.40 mL min-1 g-1) was obtained at the rate ranging from 8 to 16 mL min-1. The tissue to medium concentration ratio of propranolol, after the perfusion with 3 to 5% BSA at the rate of 8 to 16 mL min-1, was approximately 35, whereas that with 3% BSA at 4 mL min-1 was reduced to about 20. The findings suggest evidence for flow-dependent in-vitro pulmonary clearance of propranolol.     

茶多酚对家兔阳离子化牛血清白蛋白肾炎模型的治疗作用

目的 探讨茶多酚对家兔阳离子化牛血清白蛋白模型(C-BSA)肾炎的治疗作用. 方法 按文献方法复制家兔C-BSA肾小球肾炎模型,并用3种不同剂量的茶多酚 (30, 100 和 300 mg*kg-1) 给C-BSA肾小球肾炎模型兔灌胃,每日1次,连续 14 d. 结果 茶多酚不仅可以显著减少模型兔 24 h 尿蛋白分泌,降低血清肌酐和尿素氮水平,而且可以减轻肾小球病理损害,并且存在明显的量效关系. 结论 茶多酚对家兔C-BSA模型肾炎具有明显的治疗作用.     

Effects of the immunosuppressants cyclosporin A and FK 506 on exocytosis in the rat exocrine pancreas in vitro.

1. We have examined the effects of the immunosuppressive drugs cyclosporin A (CsA) and FK 506 on exocytosis in two in vitro preparations of the exocrine pancreas-lobules and dispersed acini. 2. In lobules taken from starved rats and stimulated with the secretagogue caerulein, both CsA and FK 506, given shortly before stimulation, caused a dose-dependent inhibition of amylase secretion. In lobules from rats that had been pretreated in vivo with the protease inhibitor FOY-305 to stimulate secretion maximally, both CsA and FK 506 inhibited secretion of newly synthesized proteins, whereas only FK 506 inhibited caerulein-stimulated amylase release. 3. These different effects of the immunosuppressants on amylase release were reflected in their effects on degranulation, as revealed by electron microscopy. Control acinar cells in lobules from FOY-305-treated rats were almost completely degranulated, whereas treatment with FK 506, but not CsA, caused the accumulation of zymogen granules close to the apical plasma membrane. 4. In dispersed acini, stimulated with the cholinomimetic secretagogue bethanechol, both CsA and FK 506 reduced the secretory response, to about 45% of control; IC50 values were 50 nM and 3 nM, respectively. A similar partial inhibition of exocytosis was seen in acini permeabilized with the bacterial toxin streptolysin O and stimulated with 10 microM Ca2+. 5. These results demonstrate that the immunosuppressants cause an inhibition of exocytosis in the exocrine pancreas that is both rapid in onset and potent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ciprofloxacin-loaded bovine serum albumin microspheres: preparation and drug-release in vitro.

Ciprofloxacin albumin microspheres were prepared by the spray drying technique, with bovine serum albumin as the natural biodegradable wall material. The spherical microspheres, flowed well, were organic solvent free and in the size range 1-5 microm. The drug release from the microspheres could be retarded by further thermal denaturation. The sustained-release microspheres were suitable for dry powder inhaled lung drug delivery systems.     

Divergent antibody isotype responses induced in mice by systemic exposure to proteins: a comparison of ovalbumin with bovine serum albumin.

Whereas many foreign proteins are immunogenic, only a proportion is associated commonly with allergy, having the potential to induce the quality of immune response necessary for IgE antibody production and the development of immediate type hypersensitivity reactions in the gastrointestinal and/or respiratory tracts. In the context of toxicological evaluations there is a need to identify those properties that confer on proteins the ability to provoke allergic reactions. The characteristics of antibody responses induced in BALB/c strain mice following administration of ovalbumin (OVA), a significant human allergen, have been compared with those provoked by bovine serum albumin (BSA), a protein considered to have more limited allergenic potential. Intranasal or intraperitoneal (ip) administration of BSA or OVA elicited vigorous IgG and IgG1 antibody responses. Differential IgE antibody production was observed, however, with OVA stimulating relatively high IgE antibody titres at all doses tested whereas no or low titre IgE antibody was detected following exposure to BSA. Furthermore, a differential capacity for IgG2a antibody responses was observed, with only BSA provoking high titres of this IgG subclass. The relative quality of induced responses was equivalent following administration of these proteins via mucosal (in) tissue or via a non-mucosal (ip) route of exposure. IgG2a antibody production is promoted by the type 1 cytokine interferon gamma (IFN-gamma), whereas IFN-gamma and the type 2 cell product interleukin 4 exert reciprocal antagonistic effects on IgE antibody responses. Although cytokine expression patterns were not analysed in this series of experiments, the differential IgE and IgG subclass antibody responses induced by BSA and OVA are consistent with the preferential activation of T helper (Th) 1- and Th2-type cells, respectively. These data indicate that proteins can provoke in mice characteristic antibody (IgE and IgG) isotype profiles suggestive of discrete T lymphocyte responses and that such differences may be associated with variable allergenic activity.     

Fluorometric determination of drug-protein association constants: binding of pamaquine by bovine serum albumin.

The binding of pamaquine to bovine serum albumin is accompanied by the enhancement of the fluorescence efficiency of the former but without shifting its fluorescence energy. This phenomenon was used to evaluate the stoichiometry and strength of the binding. The results indicate that three singly protonated pamaquine molecules are bound by each bovine serum albumine molecule. The individual binding constants were calculated by using the Bjerrum technique. The average values of the three constants were K1 = 6.4 X 10(7), K2 = 3.1 X 10(6), and K3 = 1.9 X 10(5), indicating that, compared to anionic drugs and fluorescent probes, pamaquine is very strongly bound by the protein.     

The effect of deuterium oxide on the conformational stability and aggregation of bovine serum albumin

Abstract

Protein aggregation is a significant problem affecting the integrity of proteins, and is a major hindrance to the development of biopharmaceutical products. Deuterium oxide (D₂O), widely used in protein characterization studies, has been shown to promote protein aggregation when used as a substitute for water in most buffered protein solutions; however, a few studies have reported minor improvements in melting point temperatures for some proteins. Our study aims to investigate the effect of D₂O on protein stability, using bovine serum albumin (BSA) as a model. We performed accelerated stability studies at high temperatures and assessed the physical and conformational stability of BSA using fluorescence spectroscopy, dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography. Our findings reveal that D₂O enhances the conformational stability of monomeric BSA, reducing monomer loss and formation of small aggregates at high temperatures. There is also an increase in the formation of larger aggregates probed by thioflavin T (ThT), however, the increase is not considered significant based on DLS results. Our findings demonstrate that exchanging water with D₂O can improve the stability of proteins in solution, by maintaining the stability of the monomeric form, which may be beneficial for the long-term storage of some biological products.     

Role of trehalose in moisture-induced aggregation of bovine serum albumin.

Moisture-induced aggregation has been identified as a key problem in the long term storage stability of therapeutic proteins. In the present work, we have investigated the impact of the disaccharide trehalose on the aggregation behavior of a model protein, bovine serum albumin (BSA) under moist conditions. About 50% aggregation of BSA was observed at a moisture level of 8mul/10mg protein. Including trehalose in the protein sample caused a significant reduction in aggregation. We address the probable mechanisms for the protective effect of trehalose by considering the various hypotheses that have been proposed in the literature. The techniques that have been used include denaturing and non-denaturing gel electrophoresis and tryptophan intrinsic fluorescence. The nature of the aggregates was studied by carrying out electrophoresis of the aggregated protein in the presence of reducing and chaotropic reagents. The interaction studies of aggregated BSA with Thioflavin T and CongoRed indicate the possibility of amyloid type of character in the former. These studies may explain the protective role of trehalose under conditions where the storage stability of therapeutic proteins is compromised.     

Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation

Protein stability is a factor limiting the use of sustained-release devices in medical applications. The aim of this study was to reduce structural perturbations occurring in the frequently used model protein, bovine serum albumin (BSA), upon microencapsulation in poly(D,L-lactide-co-glycolide) (PLG) microspheres. Spray freeze-dried BSA was encapsulated into PLG microspheres by a completely non-aqueous oil-in-oil encapsulation procedure. FTIR spectroscopy was used as a non-invasive method to quantify procedure-induced structural perturbations in BSA. Spray-freeze drying of BSA caused significant structural perturbations that were minimized by co-spray freeze-drying BSA with trehalose. BSA-containing microspheres were produced by suspension of the powder by homogenization in methylene chloride containing PLG, followed by formation of coacervate droplets by the addition of silicon oil and hardening using the solvent heptane. Resulting microspheres had dimensions of approximately 100 microm and the encapsulation efficiency for BSA was > 90%. FTIR data showed that the structure of the BSA-trehalose formulation encapsulated into PLG microspheres was less perturbed than that of BSA obtained from buffer alone. The results demonstrate that the structure-guided encapsulation approach introduced for non-aqueous casting encapsulation procedures can be extended to the non-aqueous production of pharmaceutically relevant PLG microspheres involving a complex encapsulation procedure.     

Study on the interaction of oxymatrine with bovine serum albumin

The binding reaction of oxymatrine (OMT) with bovine serum albumin (BSA) was studied by the methods of isothermal titration calorimetry, fluorescence, and circular dichroism (CD) spectroscopy. The thermodynamic results indicated that there were two classes of binding sites on the BSA molecule for OMT molecule. When the drug molecule binding to the first class of sites, the standard changes of enthalpy (ΔH ₁°) and entropy (ΔS ₁°) were (−1.07 ± 0.50) kJ/mol and (98.3 ± 0.50) J/mol/K, respectively. The possible largest number of binding site (N ₁) was (10.0 ± 0.20). This type of binding was an enthalpy–entropy synergically driven process. On the second class of binding sites, the standard changes of enthalpy (ΔH ₂°) and entropy (ΔS ₂°) were (1.91 ± 0.03) kJ/mol and (79.8 ± 0.40) J/mol/K, respectively. The possible largest number of binding site (N ₂) was (25.0 ± 0.30). This type of binding was entropy driven process. The intrinsic fluorescence of BSA was slightly quenched by the formation of BSA–OMT complex. The CD spectra experiment showed that the α-helix contents of BSA decreased. These revealed that the microenvironment and conformation of BSA were changed in the binding reaction.