Lysis protein S of phage lambda functions in Saccharomyces cerevisiae.

The lambda S lysis gene was cloned into a Saccharomyces cerevisiae expression vector under GAL1 control. Induction with galactose in S. cerevisiae terminated cell growth and prevented colony formation. Several membrane proteins immunoreactive with anti-S antibody accumulated in the membranes, indicating that sodium dodecyl sulfate-resistant oligomers of S are formed, similar to those observed in the membranes of Escherichia coli cells killed by expression of the S gene. These observations suggest that the S gene product functions as a cytotoxic protein in the yeast cytoplasmic membrane as it does in the bacterial membrane.     

Production and characterization of antibody blocking epidermal growth factor:receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 . 10(6) epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes. The immunoglobulin G fraction of this immune sera inhibited 125I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of 125I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5 degrees C). Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

The contribution of specific cytochromes P-450 in the metabolism of 7,12-dimethylbenz[a]anthracene in rat and human liver microsomal membranes

The role of specific cytochrome P-450 isoenzymes in the regio-selective metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) has been studied in microsomal membranes from rat and human liver. An antibody inhibition study using membranes from phenobarbital-treated rats demonstrates that a member(s) of the CYP2C family accounts for up to 90% of the formation of the proximate carcinogen, DMBA-3,4-diol, and makes significant contributions to the formation of DMBA-5,6-diol and DMBA-8,9-diol. In these membranes the formation of DMBA-5,6-diol can be entirely accounted by the combined activity of members of the CYP2C and CYP2B families. The metabolism of DMBA has been investigated in human using microsomes from 10 individuals and the metabolites formed by these membranes were found to be mainly hydroxymethyl- and -diol products. The rates of formation of each metabolite show considerable interindividual variation and there was no correlation between these rates for any pairing of metabolites. The CYP content in these membranes of specific members of families 1, 2, 3 and 4 did correlate with the rates of formation of individual metabolites. Surprisingly there was no correlation between the content of CYP2C and formation of DMBA-3,4-diol but an antibody to rat CYP2C6 partially inhibited the formation of this metabolite. The results indicate that in human both inducible sub-families of CYPs, particularly of the PB-type, and constitutively expressed CYPs may be important in DMBA metabolism and that each metabolite may be produced by the combined activity of several CYP isoforms.     

Production and characterization of antibody blocking epidermal growth factor: Receptor interactions

Membranes were prepared from the human epithelioid carcinoma cell line A-431 which has approx. 2 · 10⁶ epidermal growth factor receptors per cell. This membrane preparation which retained a high epidermal growth factor binding specific activity was used as an antigen to produce antisera in rabbits. Double-immunodiffusion experiments demonstrated that the immune serum contained precipitating antibodies to several components of detergent solubilized A-431 membranes.The immonoglobulin G fraction of this immune sera inhibited ¹²⁵I-labeled epidermal growth factor binding to receptors in: (1) intact human and mouse cells; (2) membrane preparations from A-431 cells and human placenta, and (3) solubilized A-431 membranes. Inhibition of ¹²⁵I-labeled epidermal growth factor binding was observed with divalent and monovalent fragments of immunoglobulin G prepared from the immunoglobulin G fraction. Also, the immunoglobulin G fraction blocked growth factor binding to membranes at low temperature (5°C).Anti-A-431 antibody blocked the induction of DNA synthesis in quiescent fibroblasts by epidermal growth factor in a manner similar to that of anti-epidermal growth factor antibody. Addition of either anti-A-431 or anti-epidermal growth factor antibodies to fibroblasts at times up to 5 h after the addition of epidermal growth factor completely reversed the hormone's mitogenic potential. At later times (after 12 h) addition of either antibody was without effect on the stimulation of DNA synthesis by epidermal growth factor. Anti-A-431 antibody did not block the induction of DNA synthesis in fibroblasts by fibroblast growth factor or serum.     

The influence of the chemical composition of cell culture material on the growth and antibody production of hybridoma cells

The multiplication and antibody production of murine hybridoma cells cultured on five different polymer membranes were tested and compared with conventional tissue culture polystyrene (TCPS). Membranes were prepared from polyacrylonitrile (PAN) and acrylonitrile copolymerized with N-vinylpyrrolidone (NVP20, NVP30), Na-methallylsulfonate (NaMAS) and N-(3-amino-propyl-methacrylamide-hydrochloride) (APMA). Cell number and antibody concentration were quantified as criteria for viability and productivity. Adhesion of hybridoma cells was characterized by vital and scanning electron microscopy. The results suggest that a strong adhesion of cells, observed on APMA and TCPS, increased cell growth but reduced monoclonal antibody production. In contrast membranes with lowered adhesivity such as NVP20 provided favourable conditions for monoclonal antibody production. In addition it was shown that this membrane also possessed a minor fouling as indicated by the low decrease of water flux across the membrane after protein adsorption. It was concluded that NVP20 could be a suitable material for the development of hollow fibre membranes for bioreactors.     

Reconstitution of the high affinity epidermal growth factor receptor on cell-free membranes after transmodulation by platelet-derived growth factor

We have prepared plasma membranes from Balb/c 3T3 fibroblasts to study the transmodulation of the high affinity epidermal growth factor (EGF) receptor. Although phorbol esters do not transmodulate the high affinity EGF receptors on these membranes, the addition of platelet-derived growth factor (PDGF) or EGF to the membranes leads to the loss of high affinity EGF binding and to the phosphorylation of several membrane proteins, including the EGF receptor. The EGF receptor is phosphorylated at tyrosine residues although we have not yet established if this represents direct phosphorylation by the PDGF receptor kinase or is mediated by activation of other cell membrane-associated tyrosine kinases. Upon treatment of the membranes with PDGF, four major phosphoproteins (of apparent molecular masses of 69, 56, 38, and 28 kDa) are released from the membrane and can be retrieved from the supernatant fluid using a reversed-phase cartridge. As assessed by immunoprecipitation with an anti-phosphotyrosine antibody, all four proteins appear to be phosphorylated on tyrosine. The time course of dissociation of these proteins from the membranes closely parallels the loss of high affinity EGF receptors. The high affinity EGF receptor can be reconstituted on PDGF-transmodulated membranes by treating the supernatant fluid with alkaline phosphatase and adding the mixture to the membranes. It appears that dephosphorylation of the released proteins is sufficient to allow reassociation with the membranes and formation of the high affinity EGF receptor complex.     

Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.     

Tau interacts with Golgi membranes and mediates their association with microtubules

Tau, a microtubule-associated protein enriched in the axon, is known to stabilize and promote the formation of microtubules during axonal outgrowth. Several studies have reported that tau was associated with membranes. In the present study, we further characterized the interaction of tau with membranous elements by examining its distribution in subfractions enriched in either Golgi or endoplasmic reticulum membranes isolated from rat brain. A subfraction enriched with markers of the medial Golgi compartment, MG160 and mannosidase II, presented a high tau content indicating that tau was associated with these membranes. Electron microscope morphometry confirmed the enrichment of this subfraction with Golgi membranes. Double-immunogold labeling experiments conducted on this subfraction showed the direct association of tau with vesicles labeled with either an antibody directed against MG160 or TGN38. The association of tau with the Golgi membranes was further confirmed by immunoisolating Golgi membranes with an anti-tau antibody. Immunogold labeling confirmed the presence of tau on the Golgi membranes in neurons in vivo. Overexpression of human tau in primary hippocampal neurons induced the formation of large Golgi vesicles that were found in close vicinity to tau-containing microtubules. This suggested that tau could serve as a link between Golgi membranes and microtubules. Such role for tau was demonstrated in an in vitro reconstitution assay. Finally, our results showed that some tau isoforms present in the Golgi subfraction were phosphorylated at the sites recognized by the phosphorylation-dependent antibodies PHF-1 and AT-8.     

Immunological Analysis of Mycoplasma Membranes

The antigens responsible for the production of antibodies to Mycoplasma laidlawii and M. gallisepticum causing growth and metabolic inhibition of these organisms were localized in the cell membrane. Various membrane fractions were tested for serological activity. Membrane lipids were completely or almost completely inactive, whereas several preparations of defatted membrane proteins retained some serological activity, shown by their ability to stimulate metabolic inhibition antibody in rabbits and to adsorb metabolic inhibition antibody and form precipitation lines with an antiserum to the membrane. When the membranes were heated to 65 C for 1 hr, they virtually lost their ability to adsorb metabolic inhibition antibody, which suggests that the antigenic determinants are proteins. Serological activity was retained in reaggregated membranes obtained by dialysis against Mg²⁺ of membranes solubilized in sodium dodecyl sulfate. The amount of solubilized membrane protein and lipid incorporated into the reaggregated membranes could be regulated by varying the Mg²⁺ concentration. As the serological tests indicated that the various membrane antigens were selectively incorporated into the different reaggregated membranes, the use of controlled reaggregation of solubilized membranes is suggested as a new tool for the fractionation and antigenic analysis of membrane proteins.     

Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.     

Regulation by intracellular Ca2+ and cyclic AMP of the growth factor-induced ruffling membrane formation and stimulation of fluid-phase endocytosis and exocytosis

Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) induce formation of ruffling membranes [T. Kadowaki et al. (1986) J. Biol. Chem. 261, 16,141-16,147] and stimulate the fluid-phase endocytosis and exocytosis [Y. Miyata et al. (1988) Exp. Cell Res. 178, 73-83] in human epidermoid carcinoma KB cells. An increase in intracellular Ca2+ concentration by treatment with A23187, a calcium ionophore, or an increase in intracellular cAMP level by treatment with dibutyryl cAMP or forskolin almost completely inhibited the insulin-, IGF-I-, or EGF-induced formation of ruffling membranes. Increases in Ca2+ or cAMP concentration also inhibited almost completely the stimulation of fluid-phase endocytosis and exocytosis elicited by these growth factors. These results suggest that the growth factor-induced ruffling membrane formation and the stimulation of fluid-phase endocytosis and exocytosis have a common regulatory mechanism involving intracellular concentrations of Ca2+ and cAMP. 125I-EGF binding assays and immunoprecipitation experiments with anti-phosphotyrosine antibody revealed that treatment of KB cells with A23187, dibutyryl cAMP, or forskolin did not inhibit the EGF binding to the cells nor subsequent tyrosine autophosphorylation of its receptors. These results indicate that Ca2+- and/or cAMP-sensitive intracellular reactions exist downstream from the receptor kinase activation in the process of these early cellular responses.     

Cellular origin and ultrastructure of membranes induced during poliovirus infection.

Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.     

Antibody-resistant spread of vesicular stomatitis virus infection in cell lines of epithelial origin.

In MDCK cells, vesicular stomatitis virus (VSV) buds exclusively from the basolateral plasma membranes beneath tight junctions, whereas influenza virus forms only at the free apical surface. Anti-VSV antiserum did not prevent the formation of plaques on MDCK cell monolayers infected with VSV, whereas plaque formation in BHK-21 cells was completely inhibited by such antiserum. Under similar conditions, homologous antiserum completely prevented plaque formation by influenza virus on MDCK cells. In several other epithelioid cell lines, VSV also formed plaques in the presence of specific antiserum. These results suggest that VSV receptors are present on basolateral membranes in the cells studied and that junctional complexes present between cells may exclude antibody from intercellular spaces and thus permit the lateral spread of virus infection in the presence of neutralizing antibody.     

Formation of the prismatic layer in the freshwater bivalve Hyriopsis cumingii: the feedback of crystal growth on organic matrix

The squeezing hypothesis and the organic frameworks preformation hypothesis propose two different mechanisms to explain the interaction between organic frameworks and crystals during biomineralization of the prismatic layer of the mollusk shell. In this study, we began to study Hyriopsis cumingii shell formation and discover that this species seemed to follow the squeezing hypothesis. During the formation of the aragonite prismatic layer in the freshwater bivalve H. cumingii, we found that crystal growth was involved in controlling initiation of formation of the interprismatic organic membranes. First, newly formed crystals were embedded in the periostracum. Next, the interprismatic organic membranes of the prismatic layer were produced via squeezing between neighboring crystals. The organic matrix secreted by the mantle continuously self‐assembled into the interprismatic organic membranes as the crystals grew. In the mature stage, the interprismatic organic membranes were shaped by crystal growth. These findings provide evidence to support the squeezing hypothesis and add to the existing knowledge about interactions that occur at the organic–inorganic interfaces during mollusk shell biomineralization.     

Antibody that neutralizes myelin-associated inhibitors of axon growth does not interfere with recognition of target-specific guidance information by rat retinal axons

During development, many CNS projection neurons establish topographically ordered maps in their target regions. Myelin-associated inhibitors of neurite growth contribute to the confinement of fiber tracts during development and limit plastic changes after CNS projections have been formed. Neutralization of myelin-associated growth inhibitors leads to an expansion of the retinal innervation of the superior colliculus (SC). In the lesioned adult mammalian CNS, these long projection neurons are usually unable to regrow axons over long distances after lesion due to myelin-associated inhibitors, which interfere with axonal growth in vivo and in vitro. Application of a specific antibody directed against myelin-inhibitors (IN-1) promotes regrowth of corticospinal tract or retinal ganglion cell axons. In the present study, we asked whether application of an antibody to myelin-associated growth inhibitors would lead to disturbances of target-specific axon guidance. To examine this issue, we used an in vitro model, the “stripe assay,” to examine the behavior of rat retinal ganglion cell axons on membranes from embryonic and deafferented adult rat SC. On membrane preparations from embryonic rat SC, retinal fibers avoid posterior tectal membranes, possibly due to the presence of a repulsive factor. Nasal retinal axons show a random growth pattern. On membranes prepared from the deafferented adult rat SC, temporal and nasal axons prefer to grow on membranes prepared from their specific target region, which suggests the involvement of target-derived attractive guidance components. The results of the present study show that retinal axons grow significantly faster in the presence of IN-1 antibody that neutralizes myelin-associated growth inhibitors present in the membrane preparations from the adult rat SC. IN-1 antibody, however, does not interfere with specific axonal guidance. This suggests that axonal guidance and specific target finding are independently regulated in retinal axons. © 1996 John Wiley & Sons, Inc.     

Activation of polyphosphoinositide phospholipase C by guanosine 5'-O-(3-thio)triphosphate and fluoroaluminate in membranes prepared from a human T cell leukemia line, JURKAT

Polyphosphoinositide hydrolysis was studied in membranes prepared from a human T cell leukemia line, JURKAT, prelabeled with myo-[2-3H]inositol. The formation of inositol bis- and trisphosphates was stimulated in a buffer with 110 nM free Ca2+ with a nonhydrolyzable GTP analogue, GTP gamma S, and NaF plus AlCl3 in a time- and concentration-dependent manner. GTP gamma S and NaF-AlCl3 had no significant effect on the inositol monophosphate level. AlCl3 enhanced the NaF-stimulated release of inositol polyphosphates. Optimum concentrations of NaF and AlCl3 produced 1.5-fold more inositol polyphosphates than that produced by optimum concentration of GTP gamma S. OKT3 monoclonal antibody, an antibody against the T-cell receptor complex, did not stimulate the inositol polyphosphate formation by JURKAT membranes even in the presence of GTP, although the antibody at the concentrations used markedly stimulated the hydrolysis of polyphosphoinositides in intact JURKAT cells.     

Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes

Spleen cells from BALB/c mice immunized with a plasma membrane-enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I-NGF to its receptor in sympathetic or sensory neurons or PC12 cells.     

The influence of 2-hydroxybiphenyl on membranes of Rhodospirillum rubrum

Summary The effects of 2-hydroxybiphenyl upon intracytoplasmic membranes of Rhodospirillum rubrum were investigated. At concentrations of 110 and 165 M of 2-hydroxybiphenyl growth was delayed, it stopped completely at a concentration of 330 M. In the latter case, instead of vesicular intracytoplasmic membranes concentric membraneous layers were found in electronmicrographs of whole cells. Inhibitor concentrations which still permit growth do not change the general appearance of intracytoplasmic membranes either in situ or in the isolated form. However, the formation of intracytoplasmic membranes was more affected by the presence of the inhibitor than was growth. Although by electron microscopy no effect of 2-hydroxybiphenyl on intracytoplasmic membranes could be revealed there were considerable influences on membrane composition. This concerned the formation of colored carotenoids and specifically the patterns of membrane proteins.Abbreviations Bchl bacteriochlorophyll - SDS Sodium dodecylsulfate     

Changes in distribution of the long form of type XII collagen during chicken corneal development.

The expression and distribution of the long form of Type XII collagen were investigated histochemically during chicken corneal development using a monoclonal antibody (P3D11) raised against the N-terminal domain of chicken Type XII collagen. Specificity of the antibody was confirmed by immunoprecipitation before and after bacterial collagenase digestion. Immunofluorescent microscopic studies showed that during chicken cornea formation, the long form of Type XII collagen is initially detected on Day 3 embryo (stage 19) in the sub-epithelial matrix of the corneal periphery and in the matrix around the optic cup. On Day 5 embryo (stage 27) the long form was expressed in the primary stroma. Thereafter, as the secondary stroma was formed, the long form localized in the sub-epithelial and sub-endothelial matrices and in the anterior region of the limbus (corneoscleral junction) before the formation of Descemet's and Bowman's membranes. After hatching, the immunoreactivity decreased predominantly in the sub-epithelial and sub-endothelial matrices but remained at the anterior region of the limbus. Immunoelectron microscopic examination demonstrated that the long form localizes in the Descemet's and Bowman's membranes and along the collagen fibrils in the stroma with a periodic repeat. Based on the distribution of the long form of Type XII collagen in the sub-epithelial and sub-endothelial matrices and limbus, it was suggested that the long form of Type XII collagen is involved in formation of the Descemet's and Bowman's membranes and in stabilization of the limbus.     

Transit of receptors for epidermal growth factor and transferrin through clathrin-coated pits. Analysis of the kinetics of receptor entry

Selective enrichment of clathrin-coated membranes by anticlathrin immunoadsorption was used to examine the internalization of receptor-ligand complexes through coated pits. Using Staphylococcus aureus-anticlathrin antibody and [35S]methionine-labeled KB cells, the kinetics of association of the epidermal growth factor (EGF-R) and transferrin receptors (TF-R) with coated membranes were directly examined. The accumulation of EGF-R in coated pits at the cell surface was dependent upon EGF binding. EGF-R then passed sequentially through a compartment which did not react with anticlathrin antibody and a second clathrin-coated compartment. The EGF-R was degraded in lysosomes with a half-life of approximately 41-55 min. The tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, appears to mimic the action of EGF in inducing EGF-R accumulation in coated pits at the cell surface and receptor internalization. In contrast to the results with EGF-R, the TF-R was found in clathrin-coated membranes in the presence or absence of TF, and the concentration of TF-R in clathrin-coated membranes did not significantly change with time. The method presented should be of great utility for examining the biochemical changes that occur during the receptor-mediated endocytosis and sorting of ligands and receptors.