An efficient process for production and purification of hyaluronic acid from <Emphasis Type="Italic">Streptococcus </Emphasis><Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Growth of Streptococcus zooepidemicus in a 10 l bioreactor with 50 g sucrose/l and 10 g casein hydrolysate/l gave 5–6 g hyaluronic acid/l after 24–28 h. Purification of hyaluronic acid gave a recovery of 65% with the final material having an Mr of ∼4 × 10⁶ Da with less than 0.1% protein.     

Synthesis of proteoglycans and hyaluronic acid by long-term cultures of testicular cells from immature and pubertal rats

Long-term cultures of somatic testicular cells derived from immature and pubertal rats were used to study the synthesis of proteoglycans (PG) and hyaluronic acid (HA). Labelled PG and HA in the culture medium, membrane-associated and intracellular pools were characterized by gel filtration, ion exchange chromatography and selected enzymatic and chemical treatments. Somatic cells synthesize a PG containing both heparan and chondroitin/dermatan sulfate (CS/DS) chains and a PG containing only CS/DS chains. No major qualitative changes in the type of PG were observed in cells derived from immature and pubertal animals. However, significant age-dependent differences in the cell distribution pattern of PG and HA were determined. This may have implications in the regulation of spermatogenesis.     

Changes in denaturation and rheological properties of collagen-hyaluronic acid scaffolds as a result of temperature dependencies

This report describes the effect of temperature on the mechanical viscoelastic properties such as: storage modulus (E′), loss modulus (E″), and loss tangent (tan δ) of the collagen sponges modified with hyaluronic acid (HA). In order to detect collagen–HA copolymer denaturation and to assess its thermal stability, the differential scanning calorimetry (DSC) supplemented by thermogravimetric (TG) measurements was used. The denaturation temperature (T_d) of unmodified collagen samples increased from 69 to 86 °C for cross-linked samples, respectively. These temperature dependencies show remarkable changes in E′ and E″ at selected temperature up to 226 °C for all samples due to the release of loosely and strongly bound water. The influence of HA on the viscoelastic behavior of collagen is manifested by a shift of the tan δ peak associated with the process of decomposition towards higher temperatures resulting in a higher thermo-stability of the modified scaffolds.     

Uptake of hyaluronan in hepatic metastases after blocking of liver endothelial cell receptors

To follow the biodistribution of exogenous hyaluronan in tumor-bearing animals, a total of seventeen inbred rats with hepatic metastases from a colonic adenocarcinoma received 125I-labelled hyaluronan by intravenous injections. Group I received only labeled hyaluronan (25 g), whereas group II received 2.5 mg chondroitin sulphate prior to labeled hyaluronan, to block receptor uptake in normal liver endothelial cells. Animals in group III received intravenous, as well as intraperitoneal chondroitin sulphate (2.5 mg), to see if a better and prolonged blocking could be achieved. Radioactivity was visualized by whole body autoradiography, using phosphorimaging and the average radioactivity determined as phosphoimaging density units of the total area of hepatic metastases, normal liver, and skeletal muscle by computer-based image analysis. At 5 h, tumors in groups II and III showed higher uptake (4.8 ±1.8, P = .01 and 3.6 ±1.1, P = .01, respectively), in comparison to group I (1.8 ±0.6), and the mean normal liver/tumor concentration ratio was reduced from 21.4 ±10.1 in group I to 5.7 ±2.7 in group II and 3.5 ±1.1 in group III (P = .008 and P = .01, respectively).Our study shows that hyaluronan targets liver metastases of a colon adenocarcinoma. Furthermore, chondroitin sulphate pretreatment increases tumor uptake, while uptake at normal receptor sites is significantly reduced. The results also suggest that after blocking of normal hyaluronan/chondroitin sulphate receptors in healthy tissue, hyaluronan may be used to deliver drugs to specific hyaluronan receptor-positive sites of pathology.     

Recent insights into microbial catalases: Isolation,production and purification

Catalase, an oxidoreductase enzyme, works as a detoxification system inside living cells against reactive oxygen species formed as a by-product of different metabolic reactions. The enzyme is found in a wide range of aerobic and anaerobic organisms. Catalase has also been employed in various analytical and diagnostic methods in the form of biosensors and biomarkers in addition to its other applications in textile, paper, food and pharmaceutical industries. New applications for catalases are constantly emerging thanks to their high turnover rate, distinct evolutionary origin, relatively simple and well-defined reaction mechanisms. The following review provides comprehensive information on isolation, production and purification of catalases with different techniques from various microbial sources along with their types, structure, mechanism of action and applications.     

链球菌合成透明质酸的研究进展

透明质酸是一种具有极大商业价值的线性、高分子粘多糖,微生物的生物合成是透明质酸来源的首选。主要综述链球菌合成透明质酸的研究概况,分析了目前存在的主要问题及解决途径,并对其前景进行了展望。     

透明质酸修饰的绿原酸脂质体对U14宫颈癌荷瘤小鼠的抑制作用

目的:对透明质酸(HA)靶向绿原酸(CA)脂质体(HA-CA脂质体)进行处方筛选,以及对U14宫颈癌小鼠的抑制作用实验。方法:筛选制备HA-CA脂质体的方法,并以磷脂比、药脂比、PBS的p H为单因素考察指标通过正交实验筛选最优处方;采用透析袋法考察HA-CA的体外释放;Bal b/c小鼠右腋皮下接种U14宫颈癌瘤株,连续尾静脉注射给药14 d后,摘取瘤体称重,并计算肿瘤生长抑制。结果:采用薄膜分散法制备脂质体,最优处方为磷脂比为4:1,药脂比为1:30,PBS的p H为7.4。HA-CA脂质体与CA脂质体释放曲线基本一致,都具有一定的缓释效果。48 h时,HA-CA脂质体和CA脂质体的累计释放度分别为78.39%、83.01%。HA-CA脂质体对U14宫颈癌小鼠的抑瘤率为60.39%,与阳性对照组环磷酰胺相当,高于CA和CA脂质体。结论:HA-CA脂质体由于其具有主动靶向配体HA的修饰,使其抑制U14宫颈癌裸鼠的效果明显高于CA和CA脂质体。     

在兽疫链球菌中表达vgb基因和HA合成基因提高透明质酸产量

透明质酸(HA)是一种在医药及化妆品领域具有广泛应用的天然粘多糖。兽疫链球菌(Streptococcuszooepidemicus)是工业上生产透明质酸的菌种之一。透明颤菌血红蛋白(VHb)具有增强细胞摄氧的作用。对生产透明质酸的兽疫链球菌进行了基因改造:将兽疫链球菌HA的合成基因hasABC以及合成透明颤菌血红蛋白的vgb基因(Vitreoscillahemoglobingene,vgb)分别或同时插入阳性菌表达质粒pEU308中,通过电转化导入兽疫链球菌中。通过一氧化碳(CO)差光谱检测到了VHb的表达。在摇瓶实验中,同时带有hasABC和vgb基因的重组菌比野生菌的透明质酸产量提高了30％。而在发酵罐中,带有这2个基因的重组菌的透明质酸产量达到了6．9g／L,高于重组菌5．5g／L的产量。实验结果表明,vgb基因的存在促进了细胞的生长,hasABC操纵子的过表达增强了透明质酸的合成。首次将VHb导入兽疫链球菌中,获得了表达,并证明其对菌体生长及透明质酸合成有促进作用。通过研究,VHb将可以在阳性菌中获得更广泛的应用。     

微生物发酵法生产高分子量透明质酸的研究进展

透明质酸是一种线性大分子黏多糖,分子量定义其流变性能,影响生理反应,并决定合适的用途。传统研究通过优化发酵提高透明质酸的产量已取得显著成效,近年来,研究重点逐渐转向如何提高透明质酸产品的分子量。目前,高分子量透明质酸具有良好的黏弹性、保湿性、黏附性,在医药中的应用是中、低分子量透明质酸不可替代的。概述了透明质酸分子量的调控机制,以及利用微生物发酵法生产高分子量透明质酸的研究进展,并对其发展方向进行了展望。     

Isolation, purification and characterization of hyaluronan from human umbilical cord residues

The main objective of this paper is to discuss new procedures of the isolation of Hyaluronan. Hyaluronic acid can be obtained from human umbilical cord residual, which is obtained from other biopharmaceutical productions. The route involves treatment of human umbilical cord residuals with sodium chloride solution, followed by ammonium quaternary salt solution precipitation; the solid is re-suspended in calcium chloride solution in order to dissociate the hyaluronan ammonium quaternary salt complex followed by ethanol-induced precipitation to give a product. The product was purified four times by chloroform extraction, and characterized by chemical methods such as the Blumenkrantz and Asboe-Hansen uronic technique for uronic acid determination, Elson Morgan qualitative tests for hexosamines, intrinsic viscosity, ion-exchange chromatography, and ¹³C NMR spectroscopy. The results showed that the product might be used in the formulation of ointment, lotion and cream for the treatment of skin diseases.     

The Effects of Hyaluronic Acid on Macrophage FC Receptor Binding and Phagocytosis Are Independent of the Mode of Depolymerization

In order to determine whether exposure of hyaluronic acid to oxygen radicals caused an alteration in its properties. independent of the change in molecular weight induced. we examined its effect upon macro-phage Fc receptor binding. High molecular weight hyaluronic acid (Healon-Pharmacia) caused a dose dependent inhibition of binding between the concentrations of 0.2–1 mg/ml. At a concentration of 0.3 mg/ml both oxygen radical depolymerized and enzymatically degraded hyaluronic acid caused an inhibition of Fc receptor binding at molecular weights of 1 × 10⁶. 1.5 × 10⁶ and 2 × 10⁶. Oxygen radical degraded hyaluronic acid caused a stimulation of Fc receptor binding at molecular weights of 2 × 10⁵ and 3.5 × 10⁵. and enzyme degraded hyaluronic acid causes stimulation at a molecular weight of 2.5 × 10⁶. Thus this “biological property” of hyaluronic acid is dependent upon molecular weight solely and not upon the mode of depolymerization.     

Systemic administration of high-molecular weight hyaluronan stimulates wound healing in genetically diabetic mice

Hyaluronic acid (HA), an essential component of the extracellular matrix, is an efficient space filler that maintains hydration, serves as a substrate for assembly of proteoglycans and is involved in wound healing. Although numerous pieces of evidence demonstrate beneficial effects in promoting wound healing in diabetes, a systemic approach has never been tested. We used an incisional wound healing model in genetically diabetic mice to test the effects of systemic injection of HA. Diabetic (n = 56) and normoglycemic (n = 56) mice were subjected to incision and randomized (8 groups of 7 animals each) to receive HA at different doses, 7.5, 15 and 30 mg/kg/i.p., or vehicle (0.9% NaCl solution) for 12 days. At the end of the experiment animals were sacrificed and skin wounds were excised for histological, biochemical and molecular analysis. Histology revealed that the most effective dose to improve wound repair and angiogenesis in diabetic mice was 30 mg/kg. Furthermore HA injection (30 mg/kg) improved the altered healing pattern in diabetic animals, increased skin remodeling proteins TGF-β and transglutaminase-II and restored the altered expression of cyclin B1/Cdc2 complex. Evaluation of skin from diabetic animals injected with HA revealed also an increase in HA content, suggesting that systemic injection may be able to restore the reduced intracellular HA pool of diabetic mice. Finally HA markedly improved skin mechanical properties. These promising results, if confirmed in a clinical setting, may improve the care and management of diabetic patients.     

凝胶层析在发酵液中分离提纯透明质酸中的应用

采用葡聚糖凝胶G-100,以0.1 mol/L氯化钠溶液为洗脱剂,在优化的凝胶层析条件(层析柱高度30 cm、进样量2 ml、洗脱流速1 ml/4 min)下,对透明质酸发酵液进行分离纯化,得到了高纯度的透明质酸.HA提取率达到79.85%,蛋白质去除率为84.93%.