Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line

Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co-transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non-transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 microg/L. By flow cytometry analysis, about 30% of the cells in the co-transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.     

Studies on nonidet P40 lysis of murine lymphoid cells. I. Use of cholera toxin and cell surface Ig to determine degree of dissociation of the plasma membrane.

Lymphoid cells from A/J mice were iodinated (125I) by the lactoperoxidase lysed with the non-ionic detergent NP-40. The plasma membrane glycolipid receptor for cholera toxin and cell surface immunoglobulin were utilized in immune precipitation systems to characterize the degree of dissociation of the plasma membrane under various conditions. It was found that at 0.1% NP-40 and at cell concentration from 5 to 10 times 10(7) cells/ml, lipid-protein and protein-lipid-protein complexes formed in NP-40 which were soluble after centrifugation at 10(5) times G. Column chromatography of 125I-cell lysates on agarose A-0.5 M in 0.1% or 0.5% NP-40/PBS indicated that the majority of iodinated cell surface material existed as aggregates in detergent micelles. The availability of the oligosaccharide moiety of the glycolipid to interact with the cholera toxin was dependent on both the detergent concentration and the cell concentration used for cell lysis. However, the cell surface immunoglobulin was immunoprecipitable under all conditions of lysis tested.     

Bioreactor culture of recombinant <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> S2 cells: characterization of metabolic features related to cell growth and production of the rabies virus glycoprotein

Although several reports have been published on recombinant protein expression using Drosophila cells, information on their metabolism and growth in vitro is relatively scarce. In the present study, we have analyzed the growth and metabolism of transfected S2 cells (S2AcRVGP) in bioreactor cultures with serum-free medium Sf900 II, to evaluate its potential for mass production of a rabies virus glycoprotein (RVGP). Cells were cultured in a 3 l-stirred-tank bioreactor at 28 °C with pH controlled at 6.2 and dissolved oxygen at 50% air saturation. The cells attained a specific growth rate and maximum cell density as high as 0.084 h⁻¹ and 2.3 × 10⁷ cell ml⁻¹, respectively. The main substrates consumed during this rapid growth phase were glucose, glutamine and proline. An atypical accumulation of ammonia and alanine was observed in the culture medium, up to 62 mM and 47 mM, respectively, but lactate was produced in low levels. After exhaustion of glutamine and proline as energy sources, alanine was consumed and production of ammonia increased. The production of recombinant RVGP reached concentrations as high as 178 μg l⁻¹. Premature exhaustion of glutamine, serine and cysteine could be related to degradation of the recombinant glycoprotein. In general, the results demonstrated that S2AcRVGP can be considered an effective vehicle for large-scale recombinant glycoprotein expression and that several critical factors of the bioprocess could be optimized to increase the quality and productivity of the RVGP.     

Rabies virus glycoprotein expression in Drosophila S2 cells. I: Design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression

The cDNA encoding the rabies virus glycoprotein (RVGP) gene was cloned in expression plasmids under the control of the inductive metallothionein promoter. They were designed in order to bear or not a secretion signal (i) and a cDNA coding for the selection hygromycin. These vectors were transfected into S2 cells, cell populations selected and subpopulations were then obtained by reselection with hygromycin. Cell cultures were examined for kinetics of cell growth, detection of RVGP mRNA and expression of RVGP. All cell populations were shown to express the RVGP mRNA upon induction. S2MtRVGPHy cell population, transfected with one vector that contains RGPV gene and selection gene, was shown to express higher amounts of RVGP as evaluated by flow cytometry (52%) and ELISA (0.64 μg/10⁷ cells at day 7). Subpopulation selection allowed a higher RVGP expression, specially for the S2MtRVGPHy⁺ (5.5 μg/10⁷ cells at day 7). NaBu treatment leading to lower cell growth and higher RVGP expression allowed an even higher RVGP synthesis by S2MtRVGPHy⁺ (8.4 μg/10⁷ cells at day 7). SF900II medium leading to a higher S2MtRVGPHy⁺cell growth allowed a higher final RVGP synthesis in this cell culture. RVGP synthesis may be optimized by the expression/selection vectors design, cell subpopulations selection, chromatin exposure and culture medium employed.     

肺炎克雷伯氏菌荚膜多糖的提取纯化及其对细胞免疫活性的影响

采用液体深层培养得肺炎克雷伯氏菌Kp9株发酵液,研究确立了菌体快速裂解条件:NP40 1%和胰蛋白酶25 0IUg菌体,50℃作用1h ,然后加入溶菌酶80μg/mL ,56℃作用1h ;裂解液经超滤浓缩和有机溶剂沉淀获得多糖粗品;多糖粗品先后经CTAB吸附分离,DEAE-SepharoseFastFlow离子交换和SephacrylS 30 0HR凝胶过滤纯化,得分子量分布相对均一的多糖纯品,产品得率为0.25 1g/L。采用淋巴细胞转化实验分别探讨了多糖粗品和纯品的免疫活性,研究显示荚膜多糖具有高效的体外细胞免疫活性,并具有典型的双向免疫调节作用。研究结论为肺炎克雷伯氏菌荚膜多糖的开发奠定了前期基础。     

One-step purification of the recombinant catalytic subunit of pyruvate dehydrogenase phosphatase

A facile one-step affinity chromatographic purification of the recombinant catalytic subunit (PDPc) of bovine pyruvate dehydrogenase phosphatase (PDP) to near homogeneity is described. PDPc binds in the presence of Ca(2+) to the inner lipoyl domain (L2) of the dihydrolipoamide acetyltransferase component (E2) of the mammalian pyruvate dehydrogenase complex. The affinity column consists of a glutathione S-transferase (GST)-L2 fusion protein bound to glutathione-Sepharose 4B beads. An extract of transformed Escherichia coli cells containing 50 mM Tris buffer (pH 7.5), 2 mM CaCl(2), 5 mM MgCl(2,) 150 mM NaCl, 0.5 mM dithiothreitol, 1% Triton X-100, and l M urea was passed through the affinity column, and the column was washed extensively with this buffer mixture. PDPc was eluted with 50 mM Tris buffer (pH 7.5) containing 5 mM MgCl(2), 0.5 mM dithiothreitol, and 1 mM EGTA. Approximately 22 mg of highly purified PDPc was obtained from 10 g (wet weight) of transformed cells. The preparation contained a small amount of a "nicked" form of PDPc. The cleavage is between Arg-394 and Arg-395.     

Comparison of chicken erythroid cell nuclear isolation methods using morphological,immunochemical and biochemical criteria

Chicken erythroid nuclei were prepared using four published methods. Our findings indicate that nuclei prepared by nitrogen cavitation are less likely to be contaminated with plasma membrane fragments than those made by procedures involving cell disruption by hypotonic lysis. However, globin gene sequences were much less sensitive to DNase I digestion in nuclei prepared by nitrogen cavitation. This suggests that the conformation of chromatin was altered by the cavitation procedure. Analysis of the proteins solubilized during limited DNase I digestion of nuclei prepared by both hypotonic lysis and cavitation revealed no appreciable differences in HMG proteins but a notable difference in the RNP-associated proteins and core histones.Abbreviations HMG high mobility group nonhistone chromosomal protein - RNP ribonucleoprotein - SSC 14 mM sodium citrate buffered saline pH 7.0 - PMSF phenylmethanesulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - DTT dithiothreitol - PBS 10 mM sodium phosphate buffered saline pH 7.2 - NP-40 Nonidet P-40 (octylphenoxypolyethoxyethanol) - SS-DNA single-stranded DNA - RSB reticulocyte standard buffer, 0.01 M NaCl, 0.003 M MgCl₂, 0.01 M Tris-HCI, pH 7.4.     

Change of hepatitis B virions (Dane particles) phosphorylation pattern by human hepatoma cell particulate fraction

Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles.     

Loss of primary alkylsulfatase and secondary alkylsulfatases (S-1 and S-2) from Pseudomonas C12B: effect of culture conditions, cell-washing procedures, and osmotic shock.

Secondary alkylsulfatases (S-1 and S-2) were detected in Pseudomonas C12B cultured in minimal media lacking alkylsulfatases. The synthesis of these enzymes was not repressed by SO4-2- and L-cysteine or derepressed by L-methionine. Growth on 4% sodium citrate caused a 98% loss in the secondary alkylsulfatase activity of cells and 9% of this activity was detected in the culture medium. Citrate (20 mM) inhibited the activity of cell extracts (18%) but the inhibition was reversible by dialysis. The primary alkylsulfatase content of cells was not diminished by growth on citrate. The MgCl2 concentration of the medium also influenced the cellular levels of secondary alkylsulfatase. Bacteria grown on MgCl2 (0.05 mM - 40 mM) exhibited progressively increasing activity while the converse distribution was observed for activity present in the medium after growth at each MgCl2 concentration. Both primary and secondary alkylsulfatases were released when cells were either subjected to osmotic shock or treated for cell wall removal. Cells washed with 0.085 M sodium citrate-10 mM Tris-20%sucrose released roughly 87% of both enzymes and MgCl2 (0.04 M) inhibited the release of primary alkylsulfatase by 11% and secondary alkylsulfatase by 50%. It is suggested that citrate chelates divalent cations necessary for the attachment of secondary alkylsulfatase at the cell periphery.     

Effects of growth medium selection on plasmid DNA production and initial processing steps

Cultures of recombinant Escherichia coli containing the plasmid pSVbeta were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria-Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.     

Factors affecting the transformation of Escherichia coli strain chi1776 by pBR322 plasmid DNA.

The susceptibility of E. coli strain chi1776 to transformation by pBR322 plasmid DNA was examined and optimized. Maximum transformation to tetracycline (Tc) resistance was achieved when cells were harvested from L broth at 5.0--6.0 . 10(7) cfu/ml, followed by washing twice in cold 0.1 M NaCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6. Cells grown in the presence of D-cycloserine (Cyc) rather than nalidixic acid (Nx) transformed markedly better. The presence of 5 mM Mg2+ ions in washing and CaCl2 solutions stimulated transformation about 2-fold. Optimal conditions for transformation included a pH range of 7.25-7.75 and a cell-to-DNA ratio of about 1.6 . 10(8) cfu/ng plasmid DNA. The frequency of transformation was highest when cells were exposed to 100 mM CaCl2 in 250 mM KCl + 5 mM MgCl2 + 5 mM Tris, pH 7.6, before mixing with DNA. A 60 min incubation period for cell + DNA mixtures held on ice produced the maximum number of Tcr transformants. In our hands, heat shocks at 37 degrees C or 42 degrees C for various times all decreased transformation to about one-half of optimal levels. Furthermore, the recovery of transformants was best when cell + DNA mixtures were plated on precooled (4 degrees C) Tc agar plates. The efficiency of plating was optimum when only 5 microliter of cell + DNA mixture was spread per plate, suggesting that non-viable background chi1776 cells on selective medium inhibited the recovery of transformants. It was also found that the presence of linear DNA molecules in cell + DNA mixtures markedly inhibited the transformation of chi1776 by pBR322 plasmid DNA. On the basis of these findings, a new procedure for the plasmid-specific transformation of E. coli chi1776 by pBR322 plasmid DNA is proposed. The use of this technique has allowed us to attain transformation frequencies in excess of 10(7) transformants/microgram pBR322 plasmid DNA.     

Decreased stability of DNA in cells treated with alkylating agents

A modified highly sensitive procedure for the evaluation of DNA damage in individual cells treated with alkylating agents is reported. The new methodology is based on the amplification of single-strandedness in alkylated DNA by heating in the presence of Mg2+. Human ovarian carcinoma cells A2780 were treated with nitrogen mustard (HN2), fixed in methanol, and stained with monoclonal antibody (MOAB) F7-26 generated against HN2-treated DNA. Binding of MOAB was measured by flow cytometry with indirect immunofluorescence. The maximal difference in fluorescence between untreated and HN2-treated cells was observed after heating at 100 degrees C for 5 min in PBS containing 1.25 mM MgCl2. Higher concentrations of MgCl2 inhibited MOAB binding to HN2-treated cells and heating at lower concentrations induced binding to control cells. Intensive binding of MOAB to control and drug-treated cells was observed after heating in Tris buffer supplemented with MgCl2. Thus, the presence of phosphates and MgCl2 during heating was necessary for the detection of HN2-induced changes in DNA stability. Fluorescence of HN2-treated cells decreased to background levels after treatment with single-strand-specific S1 nuclease. MOAB F7-26 interacted with single-stranded regions in DNA and did not bind to dsDNA or other cellular antigens. Specific reactivity of MOAB F7-26 with deoxycytidine was established by avidin-biotin ELISA. Single-stranded conformation was necessary for the binding of MOAB to deoxycytidine on the DNA molecule. It is suggested that alkylation of guanines decreased the stability of the DNA molecule and increased the access of MOAB F7-26 to deoxycytidines on the opposite DNA strand.     

Influence of culture conditions on recombinant Drosophila melanogaster S2 cells producing rabies virus glycoprotein cultivated in serum-free medium

The recombinant G glycoprotein from the surface of the rabies virus (RVGP) is a promising candidate as a rabies vaccine component and also for diagnostic purposes. In this study, RVGP production by transfected Drosophila melanogaster S2 cells cultivated in a serum-free medium (supplemented IPL-41 medium) was carried out. The effects of pH and pO₂ were evaluated in batch culture in parallel spinner flasks. The use of a pH equal to 6.3 and a pO₂ of 40% air saturation resulted in the highest RVGP content. These conditions were also used in fed-batch mode, yielding a RVGP content level of 98 g/10⁷ cells. The main nutrients consumed were glucose, glutamine, asparagine, serine and proline and the major metabolites produced were alanine and ammonia, according to the metabolism studies performed. Since RVGP is a transmembrane protein, two different methods for protein recovery were assessed and compared. Detergent-based cell disruption showed to be more effective than mechanical disruption with glass beads for glycoprotein recovery.     

Detection of Escherichia coli O157:H7 using immunomagnetic separation and absorbance measurement

An assay system for detection of Escherichia coli O157:H7 was developed based on immunomagnetic separation of the target pathogen from samples and absorbance measurement of p-nitrophenol at 400 nm from p-nitrophenyl phosphate hydrolysis by alkaline phosphatase (EC 3.1.3.1) on the "sandwich" structure complexes (antibodies coated onto micromagnetic beads--E. coli O157:H7-antibodies conjugated with the enzyme) formed on the microbead surface. The effects of immunoreaction time, phosphate buffer concentration, pH and temperature on the immunomagnetic separation of E. coli O157:H7 from samples were determined and the conditions used for the separation were 1-h reaction time, 1.0 x 10(-2) M PBS, pH 8.0 and 33 degrees C in this system. The effects of MgCl(2) concentration, Tris buffer concentration, pH and temperature on the activity of alkaline phosphatase conjugated on the immuno-"sandwich" structure complexes were investigated after immunomagnetic separation of the target pathogen and the conditions used for the enzymatic amplification were 1.0 x 10(-4) M MgCl(2), 1.0 M Tris buffer, pH 8.0, 28 degrees C and 30-min reaction time during the assay. The selectivity of the system was examined and no interference from the other pathogens including Salmonella typhimurium, Campylobacter jejuni and Listeria monocytogenes was observed. Its working range was from 3.2 x 10(2) to 3.2 x 10(4) CFU/ml, and the relative standard deviation was 2.5-9.9%. The total detection time was less than 2 h.     

In Situ Assay of Hormone-Stimulated Adenylyl Cyclase in 96-Well Microtitration Plates: An Aide to Rapid Identification of Transformed Cell Clones

Abstract

An in situ assay able to detect hormonally stimulated or inhibited adenylyl cyclase (AC) activity on as few as 5,000 cells/well has been developed. In addition the assay monitors phosphatase activity which serves as a marker for cell density. Cells are plated in replicate wells at least one day before the assay, and the medium containing AC reagents, an ATP regenerating system, a PDE inhibitor, additives that regulate receptors and/or G proteins, and 5 mM pnitrophenyl phosphate (pNPP), plus Tris buffer to pH 7.5 is added. The hypotonic medium causes permeabilization of cells without massive lysis. After stopping the reaction with 100 μl of a solution with ATP, cAMP and SDS, the color indicating phosphatase activity is quantified by an ELISA reader, and AC activity measured by standard methods. At proper cell density (pNPP hydrolysis) the assay shows proportionality up to 2 hours. The assay is particularly useful in transfection experiments. As few as 50,000 cells can be plated and identified as receptor “positive” or receptor “negative”. The assay was key to our cloning of the V2 AVP receptor. The assay accelerated the preparation of stable cell lines with LH, FSH, adrenergic and serotonin IDβ/1B and IE receptors. It should also be useful in studies in which the transfected cDNA encodes the adenylyl cyclase proper.     

Response to (chloro)biphenyls of the polychlorobiphenyl-degrader Burkholderia xenovorans LB400 involves stress proteins also induced by heat shock and oxidative stress

We report the effects of 4-chlorobiphenyl and biphenyl on the physiology, morphology and proteome of the polychlorobiphenyl-degrader Burkholderia xenovorans LB400. The exposure to 4-chlorobiphenyl decreases the growth of LB400 on glucose, and cells exhibit irregular outer membranes, a larger periplasmic space and electron-dense granules in the cytoplasm. Additionally, lysis of cells was observed during incubation with 4-chlorobiphenyl or biphenyl. Proteome of B. xenovorans LB400 exposed to biphenyl and 4-chlorobiphenyl were analysed by two-dimensional gel electrophoresis. Besides induction of the Bph enzymes of biphenyl catabolic pathways, incubation with 4-chlorobiphenyl or biphenyl results in the induction of the molecular chaperones DnaK and GroEL. Induction of these chaperones, which were also induced during heat shock, strongly suggests that exposure to (chloro)biphenyls constitutes stress conditions for LB400. During growth of LB400 on biphenyl, oxidative stress was evidenced by the induction of alkyl hydroperoxide reductase AhpC, which was also induced during exposure to H(2)O(2). 4-chlorobiphenyl and biphenyl induced catechol 1,2-dioxygenase, as well as polypeptides involved in energy production, amino acid metabolism and transport.     

E. coli biofilm formation and its susceptibility towards T4 bacteriophages studied in a continuously operating mixing – tubular bioreactor system

A system consisting of a connected mixed and tubular bioreactor was designed to study bacterial biofilm formation and the effect of its exposure to bacteriophages under different experimental conditions. The bacterial biofilm inside silicone tubular bioreactor was formed during the continuous pumping of bacterial cells at a constant physiological state for 2 h and subsequent washing with a buffer for 24 h. Monitoring bacterial and bacteriophage concentration along the tubular bioreactor was performed via a piercing method. The presence of biofilm and planktonic cells was demonstrated by combining the piercing method, measurement of planktonic cell concentration at the tubular bioreactor outlet, and optical microscopy. The planktonic cell formation rate was found to be 8.95 × 10⁻³ h⁻¹ and increased approximately four-fold (4×) after biofilm exposure to an LB medium. Exposure of bacterial biofilm to bacteriophages in the LB medium resulted in a rapid decrease of biofilm and planktonic cell concentration, to below the detection limit within < 2 h. When bacteriophages were supplied in the buffer, only a moderate decrease in the concentration of both bacterial cell types was observed. After biofilm washing with buffer to remove unadsorbed bacteriophages, its exposure to the LB medium (without bacteriophages) resulted in a rapid decrease in bacterial concentration: again below the detection limit in < 2 h.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

Studies on the function of cell surface glycoproteins. I. Use of antisera to surface membranes in the identification of membrane components relevant to cell-substrate adhesion

An antiserum prepared against purified surface membranes of transformed BHK21/C13 cells (C13/B4) reversibly rounded and detached hamster cells from plastic tissue culture plates but did not affect cells of other species. Antiserum treatment did not alter the growth rate of C13/B4 or BHK21/C13 cells; however, NIL-8 cells exposed to the antiserum detached from the substrate and stopped growing, but remained viable for up to 72 h in the presence of the antiserum. Rounding and detachment were not inhibited by DNP or cycloheximide. Antiserum-detached cells did not reattach in the presence of these inhibitors. F(ab)' fragments also induced rounding, thus ruling out the involvement of complement and ligand-induced rearrangement of surface antigens in rounding and detachment. Three different surface-reactive immunoglobulin preparations were used in indirect immunoprecipitation studies in an attempt to identify cell surface antigens involved in regulating adhesion and morphology. Antiserum against surface membranes (anti-M) and against material shed by the cells into serum-free medium (anti-SFM) caused rounding and detachment, but a third antiserum (anti-LIS) prepared against a partially purified glycoprotein did not. All three immunoglobulin preparations precipitated glycoproteins with an apparent mol wt of 120,000 daltons from a crude membrane preparation solubilized by Nonidet NP-40. The two immunoglobulin preparations that caused rounding precipitated an additional glycoprotein peak of 140,000 daltons. Extensive preabsorption of the extract with anti-LIS immunoglobulin enriched the anti-membrane and antiserum-free medium precipitates for the 140,000-dalton peak. Anti-M immunoglobulin eluted from intact cells and subsequently used to precipitate NP-40 solubilized membrane constituents also reacted with a group of glycoproteins of approximately 140,000 mol wt. Therefore, this group of glycoproteins was considered most likely to be the glycoproteins involved in substrate adhesion and maintenance of cellular morphology.