Isolation and culture of smooth muscle cells from human umbilical cord arteries

Summary A short method is described for obtaining a large number of pure vascular smooth muscle cells in culture. The smooth muscle cells were isolated from human umbilical cord arteries digested twice by an enzyme mixture of collagenase, trypsin, elastase, and DNAase with addition of α-tosyl-lysyl chloromethane. Primary cell culture and first subculture were not contaminated by endothelial cells, no Factor VIII being produced. The cultures consisted of smooth muscle cells as appeared from phase contrast and electron microscopy. Part of this study was supported by a scholarship from the Dutch Ministry of Education and Science and by the Leyden University Foundation.     

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32 000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid--clotting factor interactions.     

High-performance capillary electrophoretic analysis of hyaluronan in effusions from human malignant mesothelioma

A procedure to quantify hyaluronan in effusions from human malignant mesothelioma using a highly sensitive and reproducible high-performance capillary electrophoresis (HPCE) method is presented. Following ethanol precipitation, hyaluronan and galactosaminoglycans were degraded to Δ^4.5-dissacharides with a mixture of chondroitinases ABC and AC. Heparan sulphate and proteins/glycoproteins were separated by ultrafiltration on a Centricon 3 membrane, and hyaluronan-derived disaccharides were analysed by direct injection of the filtrate into a HPCE system. Determination of hyaluronan in effusions from five healthy individuals and three patients with mesothelioma gave values comparable to those found using the HPLC method. One of the advantages of the HPCE method as compared to HPLC is the low solvent consumption. The much lower detection limit (attomole level) of the HPCE method may also allow the analysis of hyaluronan content in serum. The contribution of HPCE in diagnosis of a neoplasm, such as human malignant mesothelioma, illustrates the great potential of this technique in the field of life sciences.     

Plasma hyaluronan concentration: no circadian rhythm but large effect of food intake in humans

This study was designed to determine if a circadian rhythm in plasma hyaluronan concentration [HA] exists in the absence of physical activity, and if plasma [HA] is associated with feeding in human subjects. Five persons were studied under standardized conditions, blood samples being taken between 0600 and 2200 hours at 30-min intervals. Any orthostatic challenge and muscle activity was abolished by immobilization by a 6° head-down bed-rest, and the effect of a quasi-continuous ingestion of energy compared a normal, three-portion diet of equivalent energy content or to fasting. Reproducibility of HA profiles on two consecutive half-days was also studied. A highly sensitive immunoassay was used to determine plasma [HA]. The data indicated that without physical activity and without food ingestion, [HA] was unchanged and displayed no diurnal rhythm. In addition, we observed that [HA] increased after the first food intake, peaking after 60 min, and concluded from our results that without ingestion of a larger meal, and sessions of postural or muscle activity, no circadian plasma [HA] rhythm exists. Accepted: 10 June 1998     

棕尾别麻蝇金属硫蛋白的分离纯化及性质分析

将棕尾别麻蝇Boettcherisca peregrina幼虫置于含800 μg/g CdCl₂的食物中取食48 h后,可诱导金属硫蛋白(MT)的合成。诱导处理后的幼虫匀浆上清液经Sephadex G-50分子筛柱、UNO^TM Q1阴离子交换柱和Bio-Gel P-6脱盐柱层析,纯化得到2个亚型,即MT-Ⅰ和MT-Ⅱ。MT-Ⅰ和MT-Ⅱ的分子量均为9 kD,每蛋白分子均含7个Cd和20个巯基,且具254 nm的Cd-SH特征吸收肩。两者的氨基酸组成中,以半胱氨酸含量最高,分别为36.6%和31.8%;而芳香族氨基酸和组氨酸含量甚少,约1%～2%。     

Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.     

A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation,purification, characterization and l-serine production

Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50 °C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40 °C for 3 h. The enzyme showed better stability under alkaline environment (pH 6.5–9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0–7.5). The SaSHMT can produce 77.76 mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate.     

羊肚菌多糖的分离纯化及组成结构分析

液体深层发酵所得的羊肚菌（Ｍｏｒｃｈｅｌｌａ ｅｓｓｕｌｅｎｔａ Ｌ．）营养液经超过滤法去掉小分子量组分,脱脂,脱色,脱蛋白质,减压低温浓缩,用超速离心法分离提纯,再用乙醇沉淀法反复多次将所要的多糖沉淀提纯,得到羊肚菌多糖（ＭＥＰ）。对其中分子量为１００００￣１０００００的多糖（ＭＥＰ－ＳＰ）,采用ＤＥＡＥ－纤维素离子交换柱,以不同浓度ＮａＣｌ为洗脱液,进行纯化分离,再经Ｓｅｐｈａｒｏｓｅ ＣＬ－     

Biology of hyaluronan: Insights from genetic disorders of hyaluronan metabolism

Hyaluronan is a rapidly turned over component of the vertebrate extracellular matrix. Its levels are determined, in part, by the hyaluronan synthases, HAS1, HAS2, and HAS3, and three hyaluronidases, HYAL1, HYAL2 and HYAL3. Hyaluronan binding proteins also regulate hyaluronan levels although their involvement is less well understood. To date, two genetic disorders of hyaluronan metabolism have been reported in humans: HYAL1 deficiency(Mucopolysaccharidosis IX) in four individuals with joint pathology as the predominant phenotypic finding and HAS2 deficiency in a single person having cardiac pathology. However, inherited disorders and induced mutations affecting hyaluronan metabolism have been characterized in other species. Overproduction of hyaluronan by HAS2 results in skin folding and thickening in shar-pei dogs and the naked mole rat, whereas a complete deficiency of HAS2 causes embryonic lethality in mice due to cardiac defects. Deficiencies of murine HAS1 and HAS3 result in a predisposition to seizures. Like humans, mice with HYAL1 deficiency exhibit joint pathology. Mice lacking HYAL2 have variably penetrant developmental defects, including skeletal and cardiac anomalies. Thus, based on mutant animal models, a partial deficiency of HAS2 or HYAL2 might be compatible with survival in humans, while complete deficiencies of HAS1, HAS3, and HYAL3 may yet be recognized.     

Expression,purification, and characterization of recombinant human H-chain ferritin

Based on their nanocage architectures, ferritins show their potential applications in medical imaging and therapeutic delivery systems. However, the recombinant human H-chain ferritin (rHF) is prone to form inclusion bodies in Escherichia coli. In our study, the cDNA of rHF was cloned into plasmid pET28a under the control of a T7 promoter. Molecular chaperones, including GroES, GroEL, and trigger factor, were coexpressed with rHF to facilitate its correct folding. The results showed that the solubility of rHF was increased more than threefold with the help of molecular chaperones. Taking advantages of its N-terminal His-tag, rHF was then purified with Ni-affinity chromatography. With a yield of 15?mg/L from bacterial culture, the purified rHF was analyzed by circular dichroism spectrometry for its secondary structure. Moreover, the rHF nanocages were characterized by transmission electron microscopy and dynamic light scattering. Our results indicate that rHF is able to self-assemble into nanocages with a narrow size distribution.     

Localization of hyaluronan in various muscular tissues

Summary The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.     

Isolation and characterization of mesenchymal stem cells from caprine umbilical cord tissue matrix

Cord tissue fills the umbilical cord around the blood vessels and contains types of stem cells (mesenchymal stem cells or MSCs) that are not generally found in cord blood. MSCs are the stem cells that give rise to many of the “support tissues” in the body, including bone, cartilage, fat and muscle. Umbilical Cord Tissue cells (UCTs) possessing the capacity to differentiate into various cell types such as osteoblasts, chondrocytes and adipocytes have been previously isolated from different species including human, canine, murine, avian species etc. The present study documents the existence of similar multipotential stem cells in caprine UCTs having similar growth and morphological characteristics. The cells were isolated from caprine umbilical cord and cultivated in DMEM (low glucose) supplemented with 15% FBS, L-glutamine and antibiotics. Primary culture achieved confluence in 5–7 days having spindle shaped morphology. The cells were morphologically homogeneous, showed robust proliferation ability with a population doubled time of 92.07 h as well as normal karyotype. In vitro self-renewal capacity was demonstrated by colony-forming unit assay (CFU). The cells expressed MSC specific markers and showed multi-differentiation capability into adipogenic and osteogeneic. The results indicated that caprine UCTs (cUCTs) were isolated and characterized from umbilical cord tissue which can be used for tissue regeneration.     

Evaluation of hyaluronan from different sources: Streptococcus zooepidemicus, rooster comb, bovine vitreous, and human umbilical cord

Sodium hyaluronate (HA) is widely distributed in extracellular matrixes and can play a role in orchestrating cell function. Consequently, many investigators have looked at the effect of exogenous HA on cell behavior in vitro. HA can be isolated from several sources (e.g., bacterial, rooster comb, umbilical cord) and therefore can possess diverse impurities. This current study compares the measured impurities and the differences in biological activity between HA preparations from these sources. It was demonstrated that nucleic acid and protein content was highest in human umbilical cord and bovine vitreous HA and was low in bacterial and rooster comb HA. Macrophages exposed to human umbilical cord HA produced significantly higher amounts of TNF-alpha relative to control or bacterial-derived HA. These results indicate that the source of HA should be considered due to differences in the amounts and types of contaminants that could lead to widely different behaviors in vitro and in vivo.     

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs’ protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.     

重组人粒细胞——巨噬细胞集落刺激因子的分离与纯化

探讨重组人粒－巨噬细胞集落刺激因子的分离、纯化工艺。实验所得工艺流程为：离收收集菌体、超声破菌、溶解复性后以３步色谱法即疏水作用色谱、离子交换色谱、凝胶过滤精制纯化,终产品过滤除菌。所得产物用ＨＰＬＣ及ＳＤＳ－ＰＡＧＥ分析,纯度大于９９％;与生白能对照测得其生物活性为１．５９＊１０＾７－１．８６＊１０＾７Ｕ／ｍｇ;产品的急性毒性实验及热原检测均符合要求。此工艺有一定的实用价值。     

Tissue structure and macromolecular diffusion in umbilical cord immobilization of endogenous hyaluronic acid

Diffusion of endogenous hyaluronic acid and ¹²⁵I-labelled albumin, monitored by desorption from umbilical cord (Wharton's jelly) slices, was studied in relation to tissue structure. Diffusion of hyaluronic acid was Fickian and some two orders of magnitude slower than that in free solution. After treatment of tissue with trypsin which removes proteoglycan(s) and degrades glycoprotein microfibrils, hyaluronic acid mobility through the collagen fibril network that remains is increased by an order of magnitude. These findings indicate that the mobility of hyaluronic acid in tissue is reduced both by the collagen network and by the presence of proteoglycan(s) and/or microfibrils. Estimates of the reduction in mobility due to physical entanglements with the fibrillar networks show that these play a major role The mobility of hyaluronic acid found for intact tissue is sufficient for it to permeate the extracellular space within its metabolic turnover. time. Labelled albumin diffusion is intact tissue, on the other hand, is reduced by only some 30% relative to free solution. This is consistent with the approximate 10% reduction found for the polysaccharide-free tissue (given by the excluded volume fraction) and the approximate 20% reduction expected for the polysaccharides in the interstitial fluid. Similar effects appear to be involved in the mobility of endogenous diffusible proteins in tissue.     

Expression, purification, and characterization of recombinant human interleukin 24 in Escherichia coli

Interleukin-24 (IL-24) can induce apoptosis of a broad range of tumor cells, and this function of IL-24 is independent of classic tumor suppressor genes, such as p53, Rb and p16. Here, we report the expression, purification and preparation of a recombinant IL-24 protein (rIL-24) without post-translational modifications, which may selectively induce apoptosis of tumor cells in vitro. We found that non-fusion rIL-24 was not able to be expressed by vectors pET11c, 28a, and 22b in Escherichia coli. To obtain recombinant non-fusion IL-24 protein, the encoding region for IL-24 was cloned between KpnI and BamHI in pET32a. The Trx (Thioredoxin)/IL-24 fusion proteins were expressed in the form of inclusion bodies in E. coli host strain BL21 (DE21). The expression level was more than 30% of total cell lysate. Inclusion bodies were disrupted, washed, and isolated at pH 9.0, and were completely dissolved in a buffer containing 2M urea at pH 9.0. After nickel ion metal affinity chromatography, gel filtration chromatography, and renaturation, the refolded fusion proteins with a purity of >96% were obtained. Trx/IL-24 proteins were digested by enterokinase (EK) to both Trx and rIL-24 fragments which then were separated by cation exchange chromatography. Cell proliferation experiments proved that the rIL-24 (98% purity) retains its cancer-selective apoptosis-inducing properties. This result suggested that the rIL-24 may have cancer therapeutic applications.