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.     

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     

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.     

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.     

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.     

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.     

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.     

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.     

Isolation,purification, and physicochemical characterization of a D-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose-Sepharose. The lectin content was about 265 mg/100g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5mM, and then p-nitrophenyl alpha-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two pI values of 5.8 and 6.1 and E(1%)(1 cm) of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS-PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca(2+) and Mn(2+) ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 degrees C for more than 90 min the lectin was fairly stable; however, when heated at 70 degrees C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 degrees C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered lambda(max) position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar D-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical beta-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.     

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.     

The distribution of renal hyaluronan and the expression of hyaluronan synthases during water deprivation in the Spinifex hopping mouse, Notomys alexis

Hyaluronan (HA) is a glycosaminoglycan that is synthesized by a family of enzymes called hyaluronan synthases (HASs), of which there are three isoforms (HAS1, 2 and 3) in mammals. The HASs have different tissue expression patterns and function, indicating that synthesis of HA and formation of the HA matrix may be regulated by various factors. The HA matrix has an important role in renal water handling and the production of a concentrated urine. We investigated the distribution of HA and the expression of HAS1, HAS2 and HAS3 mRNAs in the kidney of the Spinifex hopping mouse, Notomys alexis, a native Australian desert rodent that is reported to produce the most concentrated urine of any mammal. After periods of three, seven and fourteen days of water deprivation, the distribution of renal HA changed considerably, and there was a general down-regulation of HAS mRNA expression. It is proposed that the regulation of HA synthesis by the different HAS isoforms during water deprivation in N. alexis, could be influenced by the molecular mass of the HA chains produced by each isoform, followed by the rate at which the individual HAS produces HA.     

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。     

Xanthophylls lutein and zeaxanthin modify gene expression and induce synthesis of hyaluronan in keratinocyte model of human skin

BackgroundClinical trials report benefits of the xanthophylls lutein and zeaxanthin for skin health. Here a keratinocyte culture was used to investigate the effects of in vitro xanthophyll treatment on gene expression and biochemical pathways.MethodsWe employed the EpiDerm tissue model, Affymetrix Human Genome Array U113, bioinformatics analyses, qPCR validation and biochemical assays for glycosaminoglycans.ResultsWe discovered 176 genes were significantly (p<0.05) down-regulated (log 2FC>2) and 47 genes were significantly up-regulated. Among the down-regulated genes we validated by qPCR marked reduction in expression of peptidase inhibitors. Bioinformatic analysis of the up-regulated genes implicated biosynthetic pathways for glycosaminoglycans. We assayed but found no increase in production of sulfated glycosaminoglycans, however there was a significant increase in biosynthesis of hyaluronic acid, a non-sulfated glycan.ConclusionsThe pattern of xanthophyll-regulated genes and the resulting biochemical responses can be linked with the responses observed in clinic trials.General significanceSkin health benefits from xanthophyll supplementation and this study reveals molecular mechanisms for some of the effects.     

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.     

芦荟多糖的分离纯化及性质研究

采用水提醇沉法提取芦荟多糖,经DEAE-C32柱层析分离,Sephades G-100进一步纯化,得AⅠ、AⅡ和AⅢ三种芦荟多糖。Sephadex G-100凝胶色谱表明,AⅠ组分为均一组分,其分子量约为3.8×10~4。借助气相色谱技术,研究了芦荟粗多糖和AⅠ组分的单糖组成。另外,红外光谱鉴定芦荟多糖主要为吡喃多糖。     

Isolation and expansion of oligodendrocyte progenitor cells from cryopreserved human umbilical cord blood

Background aimsOligodendrocyte precursor cells (OPC) hold promise as a cellular therapy for demyelinating diseases. The feasibility of using OPC-based therapies in humans depends upon a reliable, readily available source. We have previously described the isolation, expansion and characterization of oligodendrocyte-like cells from fresh human umbilical cord blood (UCB). We now describe the isolation and expansion of OPC from thawed, cryopreserved UCBMethodsWe thawed cryopreserved UCB units employing a standard clinical protocol, then isolated and plated mononuclear cells under previously established culture conditions. All OPC cultures were trypsinized at 21 days, counted, then characterized by flow cytometry after fixation, permeablization and labeling with the following antibodies: anti-oligodendrocyte marker 4 (O4), anti-oligodendrocyte marker 1 (O1) and anti-myelin basic protein (MBP). OPC were also placed in co-culture with shiverer mouse neuronal cells then stained in situ for beta tubulin III (BT3) and MBP as a functional assay of myelination.ResultsThe average OPC yield per cryopreserved UCB unit was 64% of that seen with fresh UCB. On flow cytometric analysis, 74% of thawed UCB units yielded cells with an O4-expression level of at least 20% of total events, compared with 95% of fresh UCB units. We observed myelination of shiverer neurons in our functional assay, which could be used as a potency assay for release of OPC cells in phase I human clinical trials.ConclusionsOur results demonstrate that OPC can be derived reliably from thawed, cryopreserved UCB units, and support the feasibility of using these cells in human clinical trials.