The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.     

Binding of products originating from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomal membrane.

The binding of products derived from the peroxidation of liver microsomal lipids to the non-lipid constituents of the microsomes was studied. To this end arachidonic acid labelled with tritium at the positions of the double bonds was given to rats and allowed to incorporate into the membrane lipids of the liver cell. When liver microsomes containing labelled arachidonic acid were incubated aerobically in the NADPH-dependent system, a marked production of malonic dialdehyde (MDA) occurred and, concomitantly, there was a consistent release of radioactivity from the microsomes into the incubation medium. The addition of EDTA to the incubation medium prevented, to a large extent, both the MDA formation and the release of radioactivity. Chromatographic studies showed that the bulk of the radioactivity released from the incubated microsomes is not MDA. In the incubated microsomes, the radioactivity decreased in total lipids, while it increased by about 15 times in the non-lipoidal residue. A similar increase in radioactivity was seen in microsomal protein, while no increase was observed in microsomal RNA (the radioactivity was negligible in both the incubated and the non-incubated samples). It seems therefore that products originating from lipoperoxidation of arachidonic acid covalently bind to the microsomal protein. In order to investigate whether alterations similar to those observed in the in vitro peroxidation of liver microsomes could be detected in the in vivo intoxication with carbon tetrachloride, rats given labelled arachidonic acid as above, were poisoned with CCl4. Sixty minutes after poisoning, the radioactivity present in the microsomal lipids was generally lower in the intoxicated rats than in the controls, while the labelling of the non-lipoidal residue and of the protein was higher in the CCl4-poisoned rats.     

牛肾组织及其培养物的不同消化法效果评价

为了比较不同消化液及消化方式对牛肾皮质组织和培养后形成单层细胞的消化分散效果并确定最适消化液和消化方式,分别用0.25%胰蛋白酶和0.25%胰蛋白酶-0.02%EDTA两种消化液,消化牛肾皮质组织及其培养形成的单层牛肾皮质细胞。牛肾皮质组织采用热(37℃)和冷(4℃)两种消化方式;经培养形成单层的牛肾皮质细胞采用室温(25℃)消化。结果显示,用0.25%胰蛋白酶-0.02%EDTA消化液热消化牛肾组织时,分散获得牛肾皮质细胞的活细胞数、存活率、贴壁率均优于其他消化方法,差异显著(P0.05)。培养形成的单层牛肾皮质细胞用0.25%胰蛋白酶-0.02%EDTA消化液的消化速度明显快于用单一0.25%胰蛋白酶消化液的消化速度,统计学分析显示具有显著性差异(P0.01),两种消化液消化所得细胞的存活率及贴壁率前者要更高,差异显著(P0.05)。     

Flow cytometric determination of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1 in formaldehyde-fixed endothelial cell monolayers

BACKGROUND: Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS: TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS: HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS: The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.     

Cell surface sialoglycopeptide metabolism and surface glycosyl transferase activity in the Ehrlich ascites cell.

1. The in vivo incorporation of radioactivity from [3H]glucosamine into a trypsin labile, cell surface sialoglycopeptide fraction (SGP) of Ehrlich ascites cells was studied in the presence and absence of puromycin pretreatment. The results indicated a much more complete inhibition of incorporation into the surface SGP than in the average intracellular acid insoluble glycoproteins. No evidence of turnover of the carbohydrate portion of the surface SGP independent of protein synthesis could be obtained. 2. However, when intact cells were incubated with labelled uridine 5'-diphosphate-N-actely glucosamine or cytidine 5'-monophosphate (CMP)-sialic acid there was some incorporation largely into acid insoluble material, suggesting the presence of glycosyl transferase activity in the surface. Further evidence for surface activity was obtained when neuraminidase pretreatment of intact cells stimulated incorporation of labelled CMP-sialic acid sixfold and almost all of the incorporated counts could be released by subsequent neuraminidase treatment. Furthermore, a much greater proportion of the incorporated counts could be released by papain than by trypsin treatment of the intact cells. These results suggest that the surface acceptor for exogenously added CMP-sialic acid is not identical to the endogenously synthesized trypsin labile surface SGP.     

Synthesis and release of procollagenase by cultured fibroblasts.

An inactive collagenase was harvested from both serum-free and serum-supplemented fibroblast monolayer cultures in periods of active collagen synthesis. The latent collagenase did not hydrolyze collagen and did not bind the potent collagenase inhibitor alpha2-macroglobulin. Activation with trypsin imparted to the enzyme the ability to hydrolyze collagen at neutral pH in a typical manner and to form an inhibited complex with alpha2-macroglobulin. The molecular weights, determined by calibrated gel filtration, were 78,000 and 60,000 for the latent and active enzymes, respectively. The data indicate that collagenase is released from the cells in inactive form, as a zymogen.     

Studies on cell-coat macromolecules in normal and virus-transformed BHK21/C13Cells

Cells have been grown in vitro in the presence of radioactive precursors; much of the glucosamine label is incorporated into the surface material, part of it is then spontaneously released into the medium, part is readily removed by an EDTA wash, and further amounts may be released by a mild trypsin treatment or by a period of chase.The material in the EDTA was from variously labelled cultures has been fractionated by Sephadex G-200 chromatography; the excluded peak has been analysed by Sepharose 6B and DEAE-cellulose chromatography and by electrophoresis on large pore polyacrylamide gels. Its sensitivity to pronase and hyaluronidase has been determined, as well as its sugar, lipids and amino acid composition.Cell-coat material from Polyoma and Rous sarcoma virus-transformed cell lines failed to show significant differences in chromatographic properties and in sugar and amino acid composition. Minor but reproducible differences were found in the polyacrylamide electrophoresis migration patterns, when comparing glucosamine-labelled materials.     

Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung.

Determination of protein transfer across the endothelial barrier or the entire alveolar capillary membrane is critical for investigation of mechanisms leading to pulmonary edema. The purpose of this study was to evaluate Evans blue dye for determination of protein clearance across cultured bovine pulmonary artery endothelial cell monolayers and as a quantitative marker for albumin leakage to the air spaces in isolated perfused rat lungs. Evans blue dye bound tightly to albumin (EBA) as determined by lack of transfer through dialysis membranes and specific elution with albumin from a molecular exclusion column. EBA was equivalent to 125I-labeled albumin for calculation of albumin clearance rates (Calb) across intact and challenged monolayers [Calb (+ vehicle) = 0.12 microliters/min; Calb (+10 nM alpha-thrombin) = 0.47 microliters/min; Calb (+5 mg/ml trypsin) = 1.29 microliters/min]. Transfer of EBA was linear with time in both the endothelial cell monolayer model and the perfused lung. EBA was a sensitive marker for early edema in the perfused lung (before detectable weight gain) as well as for severe edema in the oxidant-injured lung (marked EBA accumulation in lavage fluid) and was a more specific marker for protein transfer than lavage fluid protein. EBA transfer is a convenient, reproducible, and accurate means to assess alterations in vascular permeability.     

人脐静脉内皮细胞的原代培养和鉴定

目的：探索建立稳定有效的脐静脉内皮细胞体外培养方法。方法：用0．1％II型胶原酶消化分离人脐静脉肉皮细胞,加入含内皮细胞生长因子的M199完全培养液中培养,用胰蛋白酶-EDTA进行消化传代培养,用光镜和免疫组化方法对培养的细胞进行形态观察和鉴定。结果：原代培养的内皮细胞在接种后24h后完全贴壁生长,第445天后融合呈铺路石样镶嵌排列,免疫组化可见胞浆中第Ⅷ因子相关抗原呈阳性反应,证实培养的细胞为内皮细胞。结论：脐静脉灌注II型胶原酶消化法配合M199完全培养液可获得高纯度的内皮细胞,细胞可传代5—6次,但细胞产出量不高,不能传10代以上,5代以后细胞形态变化较大,对于复杂的基础研究应用受限。     

Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.     

Localization of 12-hydroxyeicosatetraenoic acid in endothelial cells

Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage.     

Isolation of endothelial cells from human placental microvessels: effect of different proteolytic enzymes on releasing endothelial cells from villous tissue

Approaches for the isolation of human placental microvascular endothelial cells (HPMEC) using proteolytic enzymes have been described recently. However, the isolation procedure and enzyme composition most suitable for optimal disaggregation of placental tissue and isolation of HPMEC has not yet been established. We tested different proteolytic enzymes and enzyme mixtures for their capabilities of releasing endothelial cells from human term placental villous tissue. Best results were obtained with a mixture of collagenase/dispase/deoxyribonuclease I (0.28%/0.25%/0.01%). By adding a discontinuous Percoll gradient centrifugation step to the enzymatic dispersion, about 1 x 10(6) cells/g tissue with more than 30% von Willebrand factor (vWf)-positive cells were obtained. However, the total cell number and number of vWf-positive cells were highly dependent on the lot of collagenase used. A perfusion step prior to mincing of villous tissue did not increase the amount of vWf-positive cells. We conclude that the methods described in this study are suitable to isolate high yields of HPMEC and that the composition of the collagenase preparation is crucial to the successful release of endothelial cells from placental tissue. To obtain pure HPMEC, further separation steps, e.g., cell sorting with antibodies against endothelial specific cell surface antigens are necessary.     

Adherence of Mycoplasma gallisepticum to glass.

Attachment of washed Mycoplasma gallisepticum cells to glass was quantified with organisms in which membrane lipids were labelled with 3H. Siliconization of the test tubes decreased attachment, while centrifugation increased it. Attachment increased with temperature, decreased with increasing pH and ionic strength of the attachment mixture, but was unaffected by Ca2+, Mg2+ and EDTA. This suggests that ionic bonds, but not salt bridges, participate in the attachment process. Glycophorin, the major receptor responsible for M. gallisepticum attachment to erythrocytes, partially inhibited the attachment of the organisms to glass. However, bovine serum albumin also decreased attachment. Extensive pretreatment of the organisms with trypsin decreased their ability to attach to glass by about 35 to 40%. Trypsin and pronase failed to detach the organisms already bound to glass, suggesting that external mycoplasma cell components, other than membrane proteins, also participate in attachment of the organisms to glass.     

A specific collagenase from rabbit fibroblasts in monolayer culture

1. Explants of rabbit skin and synovium in tissue culture secreted a specific collagenase into their culture media. Primary cultures of fibroblast-like cells, which were obtained from these tissues and maintained in culture for up to 14 subculture passages, also secreted high activities of a specific collagenase into serum-free culture medium. Secretion of enzyme activity from the cell monolayer was at constant rate for over 100h and continued for up to 8 days in serum-free culture medium. The enzymic activity released was proportional to the number of cells in the monolayer. 2. The fibroblast collagenase was maximally active between pH7 and 8. At 24 degrees C the collagenase decreased the viscosity of collagen in solution by 60%. The collagen molecule was cleaved into three-quarters and one-quarter length fragments as demonstrated by electron microscopy of segment-long-spacing crystallites (measured as native collagen molecules aligned with N-termini together along the long axis), and by polyacrylamide-gel electrophoresis of the denatured products. The collagenase hydrolysed insoluble collagen, reconstituted collagen fibrils and gelatin, but had no effect on haemoglobin or Pz-Pro-Leu-Gly-Pro-d-Arg (where Pz=4-phenylazobenzyloxycarbonyl). 3. The fibroblast collagenase was partially purified by gel filtration and the molecular weight was estimated as 38000. The activity of the partially purified enzyme was stimulated by 4-chloromercuribenzoate, inhibited by EDTA, cysteine, 1,10-phenanthroline and serum, but was unaffected by di-isopropyl phosphorofluoridate, Tos-LysCH(2)Cl and pepstatin. 4. Long-term cell cultures originating from rabbit skin or synovium from rabbits with experimentally induced arthritis also secreted specific collagenase. Human fibroblasts released only very small amounts of collagenase.     

Countercurrent distribution in a 2-phase polymer system of cultured cells following their exposure to trypsin, versen, dimethyl sulfoxide and suboptimal temperature

A study was made of the effects of trypsin (0.25%) EDTA (0.02%), DMSO (10%) and suboptimal temperatures (16-18 degrees C) on the line L cells of monolayer and suspension cultures by the method of counter-current distribution in two-phase polymer system Dextran-500/poly (ethylene) glycol-6000. It has been shown that these agents cause the increase in number of cells with low partition coefficient, which is most expressed in the population of monolayer subline cells.     

Effect of dimethyladipimidate and dimethylsuberimidate on cell-cell adhesion in rat fibroblasts

In a study performed to identify the molecular mechanisms which regulate cell to cell adhesion and contact inhibition in neoplastic and syngeneic normal cells of the rat we have observed that the adhesive capacity depends on the reagents used, either EDTA or trypsin, to release the cells from monolayer. Taking profit of this last property and of the possibility of blocking free -NH2 groups on membrane proteins with specific cross-linking reagents "in vitro", we have studied in this work the behaviour of the proteins of the cell coat involved in cell to cell adhesion of rat fibroblasts FG/2. The cross-linking reagents used were dimethyladipimidate (DMA) and dimethylsuberimidate (DMS). The cells were exposed to the reagents at 0 degrees C for 30'. Cell to cell adhesion was measured by determining the percentage of single cells labeled with 3H-leucine, adhering to a confluent monolayer at different incubation times. The inhibitory effect on cell to cell adhesion brought about by cross-linking reagents indicates that a) EDTA-released cells are more sensitive to both imides than those released with trypsin, b) DMA is more effective on trypsin-released cells and c) DMS is more effective on EDTA-released cells. Therefore, we conclude that the inhibition of adhesion by reaction with the two cross-linking reagents is more likely due to a stiffening of the molecules of the cell coat involved in the adhesion, rather than to the modification of -NH2 residues which should specifically participate to adhesive process.     

牛成纤维细胞的分离与体外培养

研究了牛胎儿和成年牛皮肤组织成纤维细胞的分离、培养、纯化方法和生长特征。通过组织块贴壁培养和分离单细胞接种培养均能获得原代牛皮肤细胞。用2.5 g/L胰蛋白酶+1mmol/L EDTA和5 g/L胶原酶I联合消化牛皮肤组织较2.5 g/L胰蛋白酶+1 mmol/L EDTA消化,得到更多的单个细胞,两者之间差异极显著(P<0.01),但其死细胞比率却有较大升高;2.5 g/L胰蛋白酶+1 mmol/L EDTA消化牛胎儿组织得到的单细胞数显著高于皮肤组织消化后得到的细胞数(P<0.01),死细胞比率也高于同种酶消化的皮肤组织。分离纯化的胎儿和皮肤成纤维细胞的生长曲线都正常且相似。2.5 g/L胰蛋白酶+1 mmol/L EDTA消化贴壁细胞后死细胞率明显高于用0.5g/L胰蛋白酶+0.53 mmol/L EDTA消化的细胞(P<0.05);培养24 h后细胞贴壁率前者要明显低于后者(P<0.05)。用0.5 g/L胰蛋白酶轻度消化混杂生长的成纤维细胞和上皮样细胞,经过反复贴壁传代2～3代,可得到较纯的成纤维细胞。     

Effects of hyaluronidase, trypsin, and EDTA on surface composition and topography during detachment of cells in culture

Cultured human embryo fibroblasts (HLM18) were labeled with [3H]glucosamine and Na35SO4, and then treated with testicular hyaluronidase, trypsin, or EDTA. Macromolecular material from the surface of these cells was characterized by DEAE-cellulose chromatography and cetylpyridinium chloride precipitation while the associated morphology of cell detachment was studied by phase contrast and scanning electron microscopy. Release of surface glycosaminoglycans by testicular hyaluronidase did not cause cell rounding or detachment. EDTA did not release cell-surface components, but caused cell contraction and detachment morphologically similar to that caused by trypsin. Large amounts of cell-surface glycoproteins and glycosaminoglycans were released by trypsin. From these observations it is concluded that hyaluronic acid is not a principal adhesive agent in the attachment of cells to a substrate. It is suggested that both EDTA and trypsin may have their primary effect upon the cytoskeleton.     

Membrane alteration of trypsin-treated smooth muscle cells and penetration by antibodies to myosin

Summary Smooth muscle cells dispersed by low concentration of trypsin (0.125%) and grown in tissue culture will not bind antibodies against smooth muscle myosin added to the culture medium. These cells will attach, flatten and contract normally. When the trypsin concentration is raised to 0.25%, many of the attached cells will not flatten. Such round cells show uptake of the myosin antibody at the periphery and in the cytoplasm, using the indirect immunofluorescent technique. At a trypsin concentration of 1%, viable cells are no longer observed and all cells show uptake of myosin antibody. It is concluded from these experiments that above a crucial trypsin concentration, the membrane becomes altered sufficiently to permit the penetration of antibodies into the cell interior.     

Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.