Partial Purification of Human Lymphocyte Activating Factor

Lymphocyte Activating Factor (LAF) is a T lymphocyte stimulant released by human monocytes cultured for 18–24 hours in tissue culture medium containing 5% human serum and the non-specific immunostimulant lipopoly-saccharide. The purification of LAF is essentially the separation of low MW LAF (～13.000) from the human serum proteins required for production of the activity. Hollow fiber ultrafiltration has been found to effect a rapid separation of low MW LAF from serum proteins, but with a yield of only 20% of the original activity. Isoelectric focusing (IEF) efficiently separates LAF from all traces of human serum, resulting in a purified sample containing no measurable protein and revealing no bands on polyacrylamide gels. The IEF purified material is about 2% of the low MW activity present in the unfractionated culture medium and is highly active in the biological assay system.     

Purification of human interleukin 1

Interleukin I (IL-1) is a lymphocyte stimulant released by human monocytes cultured for 18–24 hours in tissue culture medium containing 5% serum and the non-specific immunostimulant lipopolysaccharide (LPS). Human IL-1 is found in the conditioned medium in a low molecular weight (～ 13,000) and a high molecular weight (～ 85,000) form. The high MW activity may result from the formation of a complex between IL-1 and serum constituents. During the course of purification, the low MW IL-1 activity is often recovered in a high MW form. Hollow fiber diafiltration and membrane ultrafiltration has been found to rapidly separate low MW IL-1 from all measurable protein with a yield of 4% of the original activity. The IL-1 which converts to the high MW form during the purification is recoverable, 21% of the original activity, but contains small amounts of serum proteins. Isoelectric focusing (IEF) of the low MW IL-1 resulted in a very highly purified sample which was analyzed by polyacrylamide gel electrophoresis (PAGE). Utilizing a new staining procedure which detects less than 1 ng of protein per band, the IEF-purified IL-1 revealed trace quantities ( < 1 ng) of a slowly migrating protein similar to immunoglobulin and no other bands. There were no bands which corresponded with the known electrophoretic mobility of IL-1. Since the samples applied to the gel contained significant biological activity, this result implies that human IL-1 is biologically active in picogram quantities.     

Purification and properties of human lymphocyte activating factor (LAF).

Lymphocyte-activating factor (LAF) has been shown to be produced by LPS-stimulated human adherent cells (monocytes) and peripheral leukocytes, but many non-macrophage cell lines failed to produce LAF. Other macrophage activators including latex microspheres, antigen-antibody complexes, and barium sulfate induce the production of LAF. There is a delay of 6 hr before significant amounts of LAF activity appear in the supernatant medium and maximum activity is found after 12 to 24 hr. Chromatography of concentrated crude supernatant fractions containing LAF activity on Sephadex G-100 gave two peaks of activity (approximately 85,000 and 13,000 daltons). The latter constitutes the major activity and has been purified at least 500-fold with Sephadex G-100, anion exchange, and adsorption chromatography. Optimal stimulation with LAF induces mitosis in 10% of murine thymocytes. The purified activity is sensitive to chymotrypsin and is not affected by treatment with sodium periodate, sulfhydryl reagents, and phenylmethanesulfonylfluoride. The response of thymocytes to LAF decreases with age after 10 weeks and thymocytes obtained from animals injected with cortisone or tumor-bearing animals have an increased responsiveness to LAF.     

Purification and Partial Characterization of an Antibacterial Protein Tz1

A strain (T3) of Bacillus has been screened from paddy field. It secretes large amount of antibacterialproteins which show a strong inhibiting activity against several pathogens of rice. This paper presentsa systematic study of the inhibition spectrum and characteristics of T3 proteins. Total proteins wereprecipitated with ammonium sulfate at 70% saturation from cell-free culture. One of the proteins(Tzl) was purified from the crude extracts with Sephadex G-100, DEAE52 and FPLC Superose 12columns. A single band was demonstrated in both 15% SDS-PAGE and IEF, with an apparent MWof 6,9 kd and a pI of 7.8. Its amino acid composition was analyzed and part of its sequence,determined.     

Production of nerve growth-stimulating factor(s) from chick embryo heart cells : Use of Cytodex 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Identification of the serum factor required for in vitro activation of macrophages. Role of vitamin D3-binding protein (group specific component, Gc) in lysophospholipid activation of mouse peritoneal macrophages

In vitro treatment of mouse peritoneal cells (mixture of adherent and nonadherent cells) with lysophosphatidylcholine (lyso-Pc) in 10% FCS supplemented medium RPMI 1640 results in a greatly enhanced FcR-mediated phagocytic activity of macrophages. This macrophage-activation process requires a serum factor. Fractionation studies with starch block electrophoresis of fetal calf and human sera revealed that alpha 2-globulin fraction contains a serum factor essential for macrophage activation. To identify the serum factor, human serum was precipitated with 50% saturated ammonium sulfate and fractionated on a Sephadex G-100 column. A protein fraction with a lower m.w. than albumin had the capacity to support activation of macrophages. The active serum factor in this protein fraction was analyzed by immunoabsorption by using rabbit antisera against three major proteins of human alpha 2-globulin. This active serum factor was shown to be a vitamin D3-binding protein (group specific component, Gc). By using a monoclonal anti-Gc-absorbed active column fraction of human serum, we observed no enhanced macrophage activation over the results with serum fraction-free cultivation of lyso-Pc-treated peritoneal cells. Cultivation of lyso-Pc-treated peritoneal cells in a medium containing a low concentration of purified human Gc protein (0.1 to 2.6 ng/ml) produced a greatly enhanced phagocytic activity of macrophages. When purified human Gc protein was used in a serum-free medium for stepwise cultivation of lyso-Pc-treated nonadherent cell types, a macrophage-activating factor was efficiently generated. Therefore, it is concluded that the vitamin D3-binding protein is the essential serum factor for the lyso-Pc-primed activation of macrophages.     

The relative contribution of somatomedin to the serum-stimulated growth of human fibroblasts

Culture conditions were defined allowing to demonstrate a stimulatory effect of both serum-contained and purified Somatomedin activity on incorporation of [3H]thymidine and replication of cultured normal human fibroblasts. The use of dialyzed human serum in MEM medium supplemented by 0.2 mM serine offered the necessary and sufficient culture conditions. A significant difference between normal and hypopituitary patients sera was found in their effect on the rate of [3H]thymidine incorporation (p < 0.0001) and on cell replication (p < 0.01). Purified Somatomedin-C, in MEM without serum, is a poor mitogen. Its activity was strongly enhanced by the addition of 0.1 % dialyzed serum and 0.2 mM serine without, however, exceeding the stimulatory level of 1 % whole normal serum. The requirement of concomitant presence, for optimal $\text{in}\text{vitro}$ cell growth, of different low and high MW serum components is discussed.     

Efficient extraction of proteins from woody plant samples for two-dimensional electrophoresis

Protein extraction from plant samples is usually challenging due to the low protein content and high level of contaminants. Therefore, the 2-DE pattern resolution is strongly influenced by the procedure of sample preparation. Efficient solubilization of proteins strictly depends on the chaotrope and detergent in the extraction buffer. Despite the large number of detergents that have been developed for the use in protein extraction and IEF, there is no single compound able to efficiently extract proteins from any source. Hence, optimization has to be performed for each type of sample. We tested several chaotrope/detergent combinations to achieve optimal solubilization and separation of proteins from Norway spruce [Picea abies (L.) H. Karst.] needles and European beech (Fagus sylvatica L.) leaves and roots. The same chaotrope mixture (7 M urea, 2 M thiourea) was found to be suitable for the extraction and separation of proteins from all samples. Nonetheless, the efficiency of the surfactants tested varied between samples so that optimal extraction and separation was achieved with different detergents or combination of detergents for each sample. The 2-DE separation of spruce needle proteins was optimal in a mixture of two zwitterionic detergents (2% CHAPS and 2% decyl dimethylammonio propanesulfonate). Beech proteins were best separated in buffers containing sugar-based detergents (2% n-octyl beta-D-glucopiranoside in the case of leaf samples and 2% dodecyl maltoside for the root samples). IEF was performed in buffers with the same composition as the extraction buffer except for the root proteins that were better focused in a buffer containing 2% CHAPS.     

Characterization of selenoprotein P as a selenium supply protein.

Selenium (Se) is well known to be essential for cell culture when using a serum-free medium, but not when a medium containing serum is used. This finding suggests that serum contains some usable form of Se. To identify the Se-supplier, T-lymphoma (Jurkat) cells were cultured for 3 days in the presence of human serum immunodepleted of Se-containing serum protein, selenoprotein P or extracellular glutathione peroxidase. The Se-dependent enzyme activities (glutathione peroxidases and thioredoxin reductase) and Se content within the cells markedly decreased only when cultured with selenoprotein P-depleted serum. Compared with other Se-containing proteins, the addition of purified selenoprotein P to the selenoprotein P-depleted serum or a serum-free medium was the most effective for the recovery of cellular glutathione peroxidase activity (index of Se status). These results suggest that selenoprotein P functions as a Se-supply protein, delivering Se to the cells.     

北京鸭血清HDL主要载脂蛋白的高效液相层析分离及其理化性质

用高效液相层析(HPLC)对北京鸭血清高密度脂蛋白(HDL)的所有载脂蛋白(apo)分离纯化,得五个主要apo峰,经SDS-PAGE鉴定,均为单一带;并做了IEF、糖基含量分析;对其中含量高的四个apo峰样进行了氨基酸组成测定和N-端部分氨基酸序列分析;从所做理化性质的研究,发现北京鸭血清HDL中的主要apo为:A-Ⅰ、C-Ⅲ_o、apoC-Ⅲ_s(s=1,2)、apoC's及可能的A-Ⅲ,几乎不含E和A-Ⅱ_o北京鸭HDL中apo的上述组成特点明显不同于易患动脉粥样硬化(As)的人及兔等动物,在一定程度上决定了北京鸭HDL的形成与代谢具有其特点,该特点与北京鸭不易形成As密切相关。     

Ability of human leukocytic pyrogen to enhance phytohemagglutinin induced murine thymocyte proliferation

Human leukocytic pyrogen, a monokine produced by stimulated human mononuclear phagocytes, will enhance the murine thymocyte proliferation response to phytohemagglutinin (lymphocyte activating factor (LAF) activity). During all steps of purification of human LP, pyrogenicity and LAF activity are coincidental suggesting a single identity for the two monokines. The LAF assay for human LP is highly sensitive and can detect human LP at a concentration of 10^?12M. Further experiments suggests that human LP and LAF activities could be destroyed by heating to 70 °C. Furthermore, while in vivo pyrogenicity of human LP can be blocked by ibuprofen, the in vitro LAF activity of the same molecule is unaffected by ibuprofen. Immune rabbit serum directed against human LP could also block in vitro LAF activity either by preincubation with LP or by blocking during culture.     

Anchorage-independent growth of normal human mesothelial cells: A sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum

Summary This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture (9). We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhihits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium with added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens. This work was supported by NIH grants RO1 AG02048 and RO1 CA26656 to James G. Rheinwald and by NIH postdoctoral fellowship F32 AG05303 to Paul J. La Rocca.     

Preparation of lymphocyte-activating factor from continuous murine macrophage cell lines

Lymphocyte-activating factor (LAF), a mitogen for thymocytes and T lymphocytes, is released into the culture medium by human mononuclear cells and mouse peritoneal exudate cells following treatment with various macrophage stimulants. Experiments were performed to determine if recently described mouse macrophage cell lines released LAF in response to stimulation with bacterial lipopolysaccharide. Four continuous cell lines (P388-D₁, J774, WEHI 3, and PU5-1.8) were found to release LAF in serum-free medium following endotoxin stimulation. The results of partial purification indicated that LAF obtained from cell lines had a higher molecular weight and lower isoelectric point than LAF from human mononuclear cells.     

Production of lymphocyte-activating factor (Interleukin 1) by macrophages activated with colony-stimulating factors

Murine peritoneal exudate macrophages incubated with medium conditioned by L929 cells were stimulated to produce lymphocyte-activating factor (LAF, Interleukin 1). This stimulatory activity was partially neutralized by antiserum prepared against partially purified L cell colony-stimulating factor (CSF) and comigrated upon gel filtration with the myeloproliferative activity. LAF-inducing activity of three different L cell CSF preparations, including one purified to homogeneity, was dependent upon the concentration of CSF. A minimum of 1,000 to 3,000 units of CSF activity was required to stimulate macrophagfes to produce LAF. Concanavalin A (Con A) stimulated splenic supernatants also contained CSF and LAF-inducing activities that co-eluted upon gel filtration. LAF-inducing activities co-eluting with the two Con A CSF peaks (apparent m.w. of 25,000 and 35,000) were effective at minimum dilutions containing 1,000 to 3,000 units of CSF activity correlating in potency with L cell-derived CSF. Based on these data, it is proposed that CSF, whether of L cell or lymphoid origin, not only has myeloproliferative activities but also is capable of stimulating macrophages to produce LAF.     

The microheterogeneity of rat TBG

Isoelectric focusing (IEF) of native sera from immature or adult rats and of purified or partially purified rat serum thyroid hormone-binding proteins, demonstrates that rat TBG is a microheterogeneous protein. Autoradiography and radioactivity scans of the IEF plates show that it consists of at least four main isoforms, with bands at pH 4.35, 4.45, 4.5 and 4.55. This pattern is remarkably similar to that reported for human TBG. This is the first demonstration of the polymorphism of this recently discovered major binding protein of the rat.     

Overproduction of recombinant human VEGF (vascular endothelial growth factor) in Chinese hamster ovary cells

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. VEGF165 is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human VEGF165 (rhVEGF165) protein. The production rate of the established CHO cells was over 80 mg/l of rhVEGF165 protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The rhVEGF165 protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified rhVEGF165 protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the rhVEGF165 protein in CHO cells differed from that in insect cells. The purified rhVEGF165 protein in this study was functionally active with a half-maximal effective concentration of 3.8 ng/ ml and specific activity of 2.5 x 105 U/mg.     

Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum

Recently improved culture conditions for human adult arterial endothelial and smooth muscle cells from a wide variety of donors have been used to study the effects of lipoproteins on proliferation of both cell types in low serum culture medium. Optimal growth of endothelial and smooth muscle cells in an optimal nutrient medium (MCDB 107) containing epidermal growth factor, a partially purified fraction from bovine brain, and 1% (v/v) lipoprotein-deficient serum was dependent on either high- or low-density lipoprotein. High- and low-density lipoprotein stimulated cell growth by three- and five-fold, respectively, over a 6-day period. Optimal stimulation of both endothelial and smooth muscle cell growth occurred between 20 and 60 micrograms/ml of high- and low-density lipoproteins, respectively. No correlation between the activation of 3-hydroxyl-3-methylglutaryl coenzyme. A reductase activity and lipoprotein-stimulated cell proliferation was observed. Lipid-free total apolipoproteins or apolipoprotein C peptides from high-density lipoprotein were partially effective and together with oleic acid effectively replaced native high-density lipoprotein for the support of endothelial cell growth. In contrast, apolipoproteins or apolipoprotein C peptides from high-density lipoprotein alone or with oleic acid had no effect on smooth muscle cell proliferation. The results suggest a functional role of high- and low-density lipoproteins and apolipoproteins in the proliferation of human adult endothelial and smooth muscle cells.     

Characterization of a fetal calf serum-derived molecule reactive with human natural antibodies: its occurrence in tissue culture-grown type C RNA viruses.

By means of a sensitive radioimmunoprecipitation (RIP) assay, simian sarcoma virus-simian sarcoma-associated virus (SSV-SSAV), purified from culture fluids of infected normal rat kidney (NRK) cells, was shown to acquire a surface antigen from serum used in the tissue culture medium. This antigen, which was acquired when serum from either fetal calf, horse, swine, rabbit, or chicken origin was used, accounted for a substantial portion (but not all) of the total precipitating activity exhibited by natural human antibodies for membrane-associated antigens of these viruses. By 1) alcohol precipitation, concanavalin A chromatography, and Sephadex G-150 filtration of fetal calf serum (FCS) proteins or 2) chromatography of serum proteins over a human IgG-containing immunoaffinity column, a glycoprotein of approximately 55,000 daltons has been identified which is a minor constituent of FCS (less than 0.1% of total protein) and has the antigenic capacity of whole FCS.     

Limb bud chondrogenesis in cell culture, with particular reference to serum concentration in the culture medium

When limb bud mesodermal cells of stages 23–24 chick embryos were plated at low cell density (2 × 10⁵ cells/cm²) and cultured in medium containing 10% fetal calf serum (FCS) (serum-rich medium), all cells became fibroblastic and no chondrocyte differentiation occurred in the culture. However, when cells of the same origin were cultured in a medium containing only 0.1% FCS (serum-poor medium), almost all the cells formed aggregates which developed further to form cartilage nodules. The loss of chondrogenic activity in serum-rich medium culture was irreversible: cultivation of the limb bud cells in serum-rich medium for 12 h abolished chondrogenic activity completely and these cells could not resume activity on re-cultivation in serum-poor medium. Calf, horse and chick serum at a concentration of 10% also induced the loss of chondrogenic activity in low cell density culture. Failure of chondrogenesis in serum-rich medium culture seemed to be due to the commitment of bipotential limb bud mesodermal cells to fibroblastic cells rather than to selective detachment of pre-committed chondroblasts.