蒿鳖养阴软坚方对ConA诱导小鼠肝纤维化的预防作用

探讨蒿鳖养阴软坚方对伴刀豆球蛋白A(ConA)诱导小鼠肝纤维化的预防作用.方法:94只Balb/c小鼠根据体质量采用随机区组法分为7组,正常组8只,模型组16只,蒿鳖养阴软坚方高、中、低剂量组,复方鳖甲软肝片组和秋水仙碱组各14只.采用尾静脉注射ConA制备小鼠肝纤维化模型,以蒿鳖养阴软坚方作为预防用药.采用HE染色及...     

伴刀豆球蛋白A，核糖和腺嘌呤协同促进缺血后心功能不全的恢复

急性缺血后心功能不全可能与细胞膜损害及高能化合物的合成障碍有关。用Ｗｉｓｔａｒ大鼠进行离体心脏灌注，３０ｍｉｎ全心缺血和３０ｍｉｎ再灌注导致心肌收缩力显著下降，细胞内钙含量升高和肌酸激酶漏出。用核糖（１ｍｍｏｌ·Ｌ１）和腺嘌呤（１ｍｍｏｌ·Ｌ１）进行再灌注，上述各项指标均无改善。用伴刀豆球蛋白Ａ（４０ｍｇ·Ｌ１）进行再灌汁，细胞内钙负荷减轻，肌酸激酶漏出减少，同时心肌收缩力增加，但ＡＴＰ含量无改善，用核糖，腺嘌呤和伴刀豆球蛋白Ａ联合进行再灌注，不但心肌收缩力显著升高，细胞内钙负荷减轻，肌酸激酶漏出减少，而且高能磷酸化合物含量显著恢复。实验表明，ＡＴＰ前体不能在短时间内使急性缺血后心功能不全恢复，但在伴刀豆球蛋白Ａ的协同作用下，能快速地恢复心肌收缩力和ＡＴＰ含量。     

基于伴刀豆球蛋白A的溶胶-凝胶互变糖敏感系统研究进展

伴刀球蛋白A(Con A)是一种由刀豆植物中提取的凝集素,其分子上存在与糖分子的特异性结合点,因此常应用于糖敏感给药系统中。文中首先介绍了基于Con A的溶胶-凝胶互变糖敏感系统的早期研究情况,提出早期研究普遍出现的难题,即系统中Con A流失产生的毒性以及系统糖敏感性的降低;而近年来的研究则采用将Con A固定在单体上或聚合物上试图解决此难题。文中还介绍了采用基于ConA的溶胶-凝胶互变糖敏感系统作为模拟性胰腺进行的基础性应用研究进展。     

Glucose-sensitive gel rheology of dextran-concanavalin A mixtures suitable for self-regulating insulin delivery

Aqueous concentrated plain mixtures of dextran and concanavalin A (con A) were examined for their rheological response to glucose for comparison with previously studied partially photopolymerized acrylic derivatives. Non-destructive oscillatory tests were undertaken within the linear viscoelastic range to examine the relationship between the rheometry and the stoichiometry of the interactive materials and to examine rheological parameters as affected by molecular weight, component ratio, temperature and glucose concentrations between 0 and 1% w/w. These simple formulations were studied at 1 and 10 Hz at 0.5% strain at both 20 and 37°C. A second simplified rheological test was undertaken to demonstrate gel-sol reversibility and to produce a measure of equilibria created between these gels and glucose solutions with which they are in contact. This mimics the conditions in which the gel acts as a responsive gateway in the insulin delivery device. It proved that the gels equilibrate with glucose solutions, rather than indiscriminately removing glucose. This is important in terms of producing a delivery device that can respond in a reversible, glucose concentration-dependent manner. The method used for this is capable of relative values only but provides information not obtainable from conventional rheometry.     

Pegylated nanoceria: A versatile nanomaterial for noninvasive treatment of retinal diseases

Oxidative stress induced reactive oxygen species has been implicated as the primary molecular mechanism in the pathogenesis of debilitating retinal diseases such as diabetic retinopathy, neovascularization and age-related macular degeneration. Nanoceria (cerium oxide nanoparticles) has recently received much attention, because of its superior and regenerative radical scavenging properties. This review focuses on retinal applications of nanoceria and functionalized nanoceria. Studies in animal models showed that nanoceria possess antioxidant, anti-inflammatory, anti-angiogenic, anti-apoptotic properties and preserves retinal morphology and prevents loss of retinal functions. Nanoceria have been tested in animal models of age-related macular degeneration and neovascularization and their efficacy have been shown to persist for a long time, without any collateral effects. To date, several pharmaceutical formulations of nanoceria have been developed for their prospective clinical ophthalmic applications such as chitosan coated nanoceria, nanoceria loaded into hydrogels, nanoceria embedded in wafers and contact lens and organosilane or polyethylene glycol functionalized nanoceria. Based on their nano size range, ocular permeation could be achieved to allow topical administration of nanoceria. PEGylation of nanoceria represents the key strategy to support eye drop formulation with enhanced corneal permeation, without altering chemical physical properties. Based on their excellent antioxidant properties, nano-size, safety and tolerability, PEGylated nanoceria represent a new potential therapeutic for the treatment.     

PEGylation Prevents the N-Terminal Degradation of Megakaryocyte Growth and Development Factor

Purpose. Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. Methods. Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. Results. The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDF. This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). Conclusions. PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhancedin-vitro shelf-life stability.     

Microencapsulation of PEGylated Adenovirus within PLGA Microspheres for Enhanced Stability and Gene Transfection Efficiency

Purpose Green fluorescent protein (GFP) encoding adenovirus (ADV) was surface modified with polyethylene glycol (PEG) for microencapsulation within poly(lactic-co-glycolic acid) (PLGA) microspheres with the aim of improving stability and gene transfection activity. Methods A series of PEGylated ADV (PEG-ADV) with different PEG seeding densities on the viral surface was prepared and the GFP expression efficiency of each PEG-ADV in the series determined. The physical stabilities of naked ADV and PEG-ADV were comparatively evaluated by exerting a high shear homogenization process or by exposure to low pH. Naked ADV or PEG-ADV was microencapsulated within PLGA microspheres using a water-in-oil-in-water (W/O/W) double emulsion and solvent evaporation method. In vitro cumulative ADV and PEG-ADV release profiles from PLGA microspheres were determined over a 10-day period. GFP transfection efficiencies into HeLa cells were quantified, and the relative extent of the immune response for ADV and PEG-ADV encapsulated within PLGA microspheres was analyzed using macrophage cells. Results The physical stability of PEGylated ADV was greatly enhanced relative to that of naked ADV under the simulated W/O/W formulation conditions, such as exposure to an aqueous/organic interface during high shear-stressed homogenization. PEG-ADV was also more stable than ADV at low pH. ADV and PEG-AD were both released from PLGA microspheres similarly in a sustained fashion. However, when the ADV and PEG-ADV encapsulated microspheres transfected into HeLa cells, PEG-ADV microspheres demonstrated a higher GFP gene transfection efficiency than ADV microspheres. The PEG-ADV microspheres also exhibited a reduced extent of innate immune response for macrophage cells. Conclusions PEGylated ADV could be more safely microencapsulated within PLGA microspheres than naked ADV due to their enhanced physical stability under the harsh formulation conditions and acidic microenvironmental conditions of the microsphere, thereby increasing gene transfection efficiency.     

Effects of CuCl2 on the dimer-tetramer equilibrium of concanavalin A

Tetrameric concanavalin A at neutral pH dissociated into the dimer when CuCl₂ was added in a concentration range comparable to protomer concentration. This effect of CuCl₂ was largely suppressed when NaCl concentration was increased. Neither the conformation of the protein nor its binding activity to 4-methylumbelliferyl α-D-man-nopyranoside was affected on addition of CuCl₂.     

聚乙二醇化蛋白质阳离子树脂色谱行为的研究

以溶菌酶(LYS)作为模式蛋白,对聚乙二醇化蛋白质在不同阳离子交换填料中色谱行为进行了比较研究。考察了不同分子质量的聚乙二醇化溶菌酶(PEG-LYS)在4种阳离子树脂中的动态载样量的差异,同时比较了不同上样量对纯度和洗脱盐浓度的影响。研究结果显示PEG修饰能降低蛋白质和离子交换树脂的结合力,且随着PEG的分子质量的增加,PEG-LYS动态载样量降低。对相同分子质量的PEG-LYS,产物纯度以及PEG-LYS的洗脱盐浓度随着上样量的增加而降低,研究结果显示,PEG分子质量和上样量对纯化工艺的研究有重要意义。     

亲和层析介质性能分析及其在单抗纯化中的应用

对6种商品化蛋白A亲和层析介质进行了性能分析,包括动态载量、洗脱条件等,并对含有单抗药物Mab-HS006的实际料液进行了捕获分离,对宿主细胞蛋白及蛋白A的残留进行了分析。结果表明,Prosep Ultra Plus和Mabselect SuRe亲和介质的载量最高,而Protein A Sepharose CL-4B和r-Protein A Sepharose的洗脱条件最温和。一步层析捕获分离Mab-HS006单抗的结果表明,各种介质的纯度均可达90%以上,Prosep Ultra Plus的宿主细胞蛋白残留最高,Mabselect SuRe蛋白A的脱落最低。     

Denaturation of concanavalin A by urea at acid pH

The denaturation of dimeric concanavalin A induced by urea at pH 3 has been studied using optical activity and sedimentation velocity. Under the conditions employed Mn⁺² and Ca⁺² are dissociated from the protein, but the basic structural elements are little changed from those prevailing in the functional lectin at pH 5.5 [H.E. Auer and T. Schilz, preceding paper in this issue]. The protein passes through three stages as the urea concentration is varied from 0 to 10 M. Below 4M urea the only effect observed is the loss of optical activity of the aromatic amino acid residues. At 4 M, a conformational change occurs producing extensive aggregation, which persists to 7 M. At 8–10 M urea a disordered monomeric protein molecule prevails. The protein could be reactivated provided that dilution to native conditions was very rapid and the protein concentration remained very low. Kinetics of denaturation were monitored by optical activity at 218, 225 and 283 nm, Transients with one, two or three components were observed, which were resolved by nonlinear regression according to sequential first-order decay laws. First order character was confirmed by independence of the kinetic parameters from protein concentration over a two- to four-fold range. Enthalpies and entropies of activation for the various steps were also determined. The transients at the three wavelengths monitor changes in β structure, β turns and aromatic groups, respectively. The urea dependence of the rate constants is unique in most cases. It is concluded that different structural elements of the concanavalin A molecule unfold independently from one another.     

Solvent Selection for Insoluble Ligands,a Challenge for Biological Assay Development: A TNF-α/SPD304 Study

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A Solubility and Related Physicochemical Property Comparison of Buprenorphine and Its 3-Alkyl Esters

Pharmaceutical Research -     

8-(3-Isothiocyanatostyryl)caffeine Is a Selective, Irreversible Inhibitor of Striatal A(2)-Adenosine Receptors

8-(3-Isothiocyanatostyryl)caffeine (ISC) was synthesized and shown to inhibit selectively the binding of [(3)H]CGS 21680 (an A(2a)-selective agonist) at adenosine receptors in striatal membranes. The K(i) value at A(2a)-receptors was found to be 110 nM (rat), with selectivity ratios for A(2a) versus A(1)-receptors in rat, guinea pig, bovine, and rabbit striatum of >100-fold. Preincubation of membranes with ISC caused a dose-dependent, irreversible antagonism of the binding of [(3)H]CGS 21680, with an IC(50) value of 3 μM. The irreversibility is likely due to the presence of the chemically reactive isothiocyanate group, since the binding of the corresponding analogue in which the isothiocyanate was replaced with a chloro group was completely reversible. The potency of ISC to irreversibly inhibit the binding of [(3)H]CGS 21680 in several species varied in the order rat ≈ guinea pig > bovine ≈ rabbit. In all four species, binding of the A(1)-selective agonist [(3)H]R-N(6)-phenylisopropyladenosine was not diminished by pre-treatment with 2 μM ISC. The kinetics of irreversible inhibition of rat A(2a)-receptors by 2 μM ISC gave a t(1/2) of approximately 3 min. Following partial inactivation, the remaining rat A(2a)-binding sites retained the same K(d) value as in control membranes for saturation by [(3)H]CGS 21680. Thus, ISC appears to be a selective affinity label for A(2a)- versus A(1)-receptors in the brain.     

Synthesis and Evaluation of a Series of 3,4,5‐Trimethoxycinnamic Acid Derivatives as Potential Antinarcotic Agents

A series of 3,4,5‐trimethoxycinnamic acid derivatives was prepared and evaluated for antinarcotic effects on morphine dependence in mice and binding affinities on serotonergic receptors. The key synthetic strategies involve generation of ketones 6–7 , esters 9–12 through condensation reaction, and amides 13–19 via coupling reaction using 1‐hydroxybenzotriazole/ethyl(dimethylaminopropryl)carbodiimide system in high yield. We found that the naloxone‐induced morphine withdrawal syndrome was significantly suppressed by new synthetic 3,4,5‐trimethoxycinnamic acid derivatives (20 mg/kg/day). Most of 3,4,5‐trimethoxycinnamic acid derivatives were found to have high affinity to 5‐HT_1A receptor. The naloxone‐induced morphine withdrawal syndrome was attenuated by (+)8‐OH‐DPAT (0.1 mg/kg/day, i.p.), a 5‐HT_1A receptor agonist. In cortical neuronal cells, (+)8‐OH‐DPAT (1 μm ) produced an elevation of the pERK 1/2 expression, and the elevated pERK levels were inhibited by WAY 100635, a 5‐HT_1A receptor‐specific antagonist. Interestingly, the pERK levels were increased by the 3,4,5‐trimethoxycinnamic acid derivatives and the derivatives‐mediated changes in pERK levels were blocked by the WAY 100635. These results suggested that new synthetic 3,4,5‐trimethoxycinnamic acid derivatives have a potential antinarcotic effect through acting as a 5‐HT_1A receptor agonist in mice.     

Characterization of salicylate binding to synovial fluid and plasma protein in patients with rheumatoid arthritis

Summary Protein binding of salicylate in synovial fluid and plasma from patients with rheumatoid arthritis was studied by equilibrium dialysis. Protein binding in the synovial fluid was considerably lower at all salicylate concentrations studied (0.07 – 2.2 mM). Scatchard plots of the data were analyzed assuming binding to two classes of binding sites, each plasma sample being diluted to an albumin concentration equal to that in synovial fluid from the same patient. Binding to the primary binding sites was considerably decreased in synovial fluid in comparison with plasma. The affinity of the secondary binding sites was slightly lower. Thus, at a low therapeutic drug concentration, the decreased binding of salicylate to synovial fluid protein in patients with rheumatoid arthritis could mainly be accounted for by decreasing affinity of binding to the primary binding sites.     

双环醇对刀豆蛋白A引起肝损伤小鼠肝脏基因表达谱的影响

本实验研究双环醇对刀豆蛋白A(concanavalin A，Con A)静脉注射引起免疫性肝损伤小鼠肝脏基因表达谱变化的影响，探讨双环醇肝保护作用的分子机制。小鼠于注射Con A 26.5 mg·kg^-1前24、 8及1 h分别口服双环醇250 mg·kg^-1。测定血清丙氨酸转氨酶(alanine aminotransferase，ALT)及天冬氨酸转氨酶(aspartate aminotransferase，AST)水平，提取小鼠肝脏总RNA，经反转录用Cy3-dUTP和Cy5-dUTP分别标记制备cDNA探针。将cDNA探针与BiostarM-40S小鼠基因表达谱芯片进行杂交，经ScanArray 4000扫描仪扫描芯片并用GenePix Pro 3.0软件进行分析。双环醇可显著抑制刀豆蛋白A引起的血清ALT和AST升高。与刀豆蛋白A对照组相比，双环醇给药组有287条基因发生差异表达，占芯片基因总数的7.00%。其中166条基因表达量明显下调，121条基因表达量明显上调。表达变化的基因主要涉及代谢与细胞色素P450、应激与炎症凋亡、细胞周期调控、信号传导以及再生等相关功能。双环醇对刀豆蛋白A引起小鼠肝损伤肝脏基因表达谱变化具有一定的影响，此结果对今后深入研究双环醇的肝脏保护作用特点和临床应用具有重要意义。     

Fatty acid and drug binding to a low-affinity component of human serum albumin,purified by affinity chromatography

Binding equilibria for decanoate to a defatted, commercially available human serum albumin preparation were investigated by dialysis exchange rate determinations. The binding isotherm could not be fitted by the general binding equation. It was necessary to assume that the preparation was a mixture of two albumin components about 40% of the albumin having high affinity and about 60% having low affinity. By affinity chromatography we succeeded in purifying the low-affinity component from the mixture. The high-affinity component, however, could not be isolated. We further analyzed the fatty acid and drug binding abilities of the low-affinity component. The fatty acids decanoate, laurate, myristate and palmitate were bound with higher affinity to the mixture than to the low-affinity component. Diazepam was bound with nearly the same affinity to the low-affinity component as to the albumin mixture, whereas warfarin was not bound at all to the low-affinity component.     

PEGylation of Octreotide: II. Effect of N-terminal Mono-PEGylation on Biological Activity and Pharmacokinetics

Purpose. To determine the optimal polyethylene glycol (PEG)-conjugate of octreotide by evaluating the effects of PEGylation chemistry on the biological activity and pharmacokinetic properties.Methods. Octreotide was chemically modified by reaction with succinimidyl propionate monomethoxy PEG (SPA-mPEG, molecular weight 2000) or succinimidyl butyraldehyde-mPEG (ALD-mPEG, molecular weight 2000 and 5000). The structural conformation of PEG-octreotides was evaluated by circular dichroism (CD), the biological activity was assessed by measuring the decrease of serum insulin-like growth factor-I levels in rats, and a pharmacokinetic study was performed after subcutaneous administration in rats. The stability against acylation was investigated with poly(d,l-lactide-co-glycolide) (PLGA).Results. ALD-mPEG was site-specific in PEGylating octreotide at the N-terminus. The mono-PEG-octreotides prepared with ALD-mPEG (mono-ALDPEG-octreotide), which alkyl bond preserves the amines positive charge, showed complete preservation of biological activity, whereas the PEG-octreotides prepared with SPA-mPEG showed lower activity. In the CD analysis, the spectra of the mono-ALDPEG-octreotides were nearly superimposable with that of native octreotide. The mono-ALDPEG-5K-octreotide showed significantly improved pharmacokinetic properties compared with mono-ALDPEG-2K-octreotide as well as native octreotide. Both mono-ALDPEG-2K- and mono-ALDPEG-5K-octreotides were stable against acylation by degrading PLGA.Conclusions. The mono-PEGylation of octreotide at N-terminus with ALD-mPEG produced a conjugate that is biologically and structurally active and stable against acylation by PLGA, and therefore it may serve as a candidate for somatostatin microsphere formulations.