ATP analogues induce membrane permeabilization in transformed mouse fibroblasts

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5′-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5′-[β,γ-methylene]triphosphate (p[CH₂]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (Δψ), determined according to the distribution of the cation tetraphenylphosphonium (TPP⁺) between the cells and the medium. Reduction of 26, 18 and 13 mV in Δψ was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH₂]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of Δψ, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the β,γ-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that Δψ is involved in the control of membrane permeability.     

ATP analogues induce membrane permeabilization in transformed mouse fibroblasts

The mechanism underlying ATP-induced permeabilization of transformed mouse fibroblasts was studied by using nonhydrolyzable analogues of ATP. Incubation of 3T6 cells with 0.6 mM of either ATP, 5'-adenylyl imidodiphosphate (p[NH]ppA) or adenosine 5'-[beta, gamma-methylene]triphosphate (p[CH2]ppA) resulted in an increase of 17-, 8- or 5-times, respectively, in the cell membrane permeability, measured by the efflux of normally impermeant metabolites from the cells. The induced cell permeabilization was preceded by a reduction in the membrane potential (delta psi), determined according to the distribution of the cation tetraphenylphosphonium (TPP+) between the cells and the medium. Reduction of 26, 18 and 13 mV in delta psi was exerted by 0.6 mM of either ATP, p[NH]ppA or p[CH2]ppA, respectively. In 3T3 cells the untransformed counterparts of 3T6 cells, neither reduction of delta psi, nor alterations in membrane permeability were exerted by either ATP or by its analogues. The data indicate that the dissociation of the beta, gamma-phosphate bond is not essential for membrane permeabilization by external ATP, implying that the binding of ATP to the cell surface of transformed cells is sufficient to initiate the permeabilization process. The data also suggest that delta psi is involved in the control of membrane permeability.     

绿色荧光蛋白基因在转基因小鼠体内的表达

建立绿色荧光蛋白（GFP）转基因小鼠,继而传代建系。采用显微注射法,将GFP基因注入FVB／NJ小鼠受精卵原核内,获得子代鼠。分娩后3周剪取仔鼠尾,提取基因组DNA,应用PCR、Southern印迹技术进行整合检测。结共用雌性小鼠200只,注射受精卵1586枚,移植卵数386枚,受体鼠32只,怀孕鼠4只,子代鼠18只,有4只为阳性：取2只首建鼠的胚胎,在荧光显微镜下观察GFP表达明显,表明初步获得了转绿色荧光蛋白基因小鼠,     

Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1.

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.     

Mechanism for selective secretion of a lysosomal protease by transformed mouse fibroblasts

Studies in recent years have indicated that secretion of certain lysosomal hydrolases can be enhanced under various conditions. One such protein, the major excreted protein (MEP) of Kirsten virus-transformed NIH 3T3 (KNIH) fibroblasts, is a lysosomal cysteine protease whose synthesis and secretion are affected by viral transformation and growth factors. We have been studying the synthesis and transport of MEP in order to understand better the mechanisms responsible for regulation of lysosomal enzyme secretion. Synthesis of MEP in KNIH cells was found to be 25-fold greater than that in untransformed NIH cells, and 94% of the MEP made was secreted. This was in contrast to NIH cells which secreted only 11% of the newly synthesized MEP. The high level of secretion by the transformed cells was relatively specific in that most other lysosomal enzymes were retained. MEP isolated from both NIH and KNIH cells exhibited a low intrinsic affinity for the mannose-6-phosphate receptor which was at least 10-fold lower than that of other lysosomal enzymes. On the basis of these results, we suggest that both the high level of MEP synthesis and the intrinsic low affinity of MEP for the receptor are responsible for the specific increase in MEP secretion by transformed cells.     

Permeability change in transformed mouse fibroblasts caused by ionophores,and its relationship to membrane permeabilization by exogenous ATP

Summary Electrogenic ionophores have been found to induce membrane permeabilization in Swiss mouse 3T3 cells that had undergone spontaneous transformation (3T6 cells). Cells attached to plastic dishes were loaded with [³H] uridine, and then the medium was replaced by buffered salt solution at pH 7.8. The enhancement of membrane permeability was assayed by following the efflux of uridine nucleotides, normally impermeant substances. Titration with electrogenic ionophores, such as carbonylcyanidem-chlorophenylhydrazone (CCCP), SF-6847 and gramicidin D, markedly increased the membrane permeability within a very narrow range of ionophore concentration. Nonelectrogenic ionophores, such as monensin and nigericin, did not affect membrane permeability. Measurements of the distribution of the lipophilic cation tetraphenylphosphonium (TPP⁺) between the cells and their environment implied that the remarkable increase in permeability took place within a narrow range of membrane potential (). The data could be explaine by a threshold value, under which aqueous channels are opened in the plasma membrane. The effects exerted by electrogenic ionophores on the plasma membrane were found to be similar to those induced by exogenous ATP. In both cases rapid efflux of K⁺, influx of Na⁺ and reduction of preceded membrane permeabilization to low molecular weight, charged molecules, such as nucleotides. It is suggested that dissipation of induces conformational alterations in membranal components, and/or topological changes, such as aggregation of protein molecules, to form membranal aqueous channels. Electrogenic ionophores permeabilize both normal (3T3) and transformed (3T6) mouse fibroblasts, whereas ATP effects are specific for transformed cells. Thus, it is postulated that ATP actsvia specific sites on the surface of transformed cells.     

Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts

Fibroblasts as well as several other cell types, secrete a number of protease inhibitors into their culture media. Among these inhibitors are the protease nexins, a class of proteins which covalently bind serine proteases, thereby inactivating their specific targets. Protease nexin-I, first discovered in human foreskin fibroblasts, binds thrombin, plasmin, and urokinase with high affinity, forming covalently linked complexes. Human fibroblasts bind complexes of protease nexin-I and its target protease via a cell-surface, high-affinity receptor. We have analyzed a number of characteristics of this receptor, and found them to be typical of class II receptors in general. At 4 degrees C binding of PN-I:protease complexes was competed by heparin. In addition, binding was independent of the particular protease bound to the PN-I; purified complexes of PN-I with thrombin or urokinase competed equipotently for [125]I-thrombin:PN-I binding. As the pH of the binding buffer was lowered, binding to cells increased. A twofold increase in binding was attained by lowering the pH from 7.5 to 4.5. This phenomenon was not due to irreversible, pH-induced changes to either the cell surface or the labeled complexes. At 37 degrees C, the removal of labeled complexes from culture medium was rapid; approximately 80% was removed by 4 hours under given conditions. The internalization of complexes was also very rapid, with an estimated ke (endocytic rate constant) of 1.0 min-1. At neutral pH, fibroblasts bind complexes in a saturable manner. Scatchard analysis yields a receptor number of 250,000 per cell and a Kd of 1 nM.     

Purification of protease nexin II from human fibroblasts

Normal human fibroblasts secrete a protein named protease nexin II (PN II) which previously was shown to form sodium dodecyl sulfate (SDS)-stable complexes with epidermal growth factor-binding protein (EGF-BP). These complexes then bind to the same cells and are rapidly internalized and degraded (Knauer, D.J., and Cunningham, D.D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2310-2314). Here we describe a procedure for purifying PN II to apparent homogeneity from serum-free culture medium conditioned by human fibroblasts. The first step employed dextran sulfate-Sepharose affinity chromatography. Further purification was achieved by ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-400. Sequence analysis of purified PN II identified 33 amino-terminal amino acids; a computer search of several protein sequence data banks failed to reveal homologies with other reported amino acid sequences. Purified PN II had an apparent Mr of 106,000 and an isoelectric point of approximately 7.2. It retained full activity after incubation in the presence of 0.05% SDS or at a pH of 1.5. PN II formed SDS-stable complexes with EGF-BP, the gamma subunit of 7 S nerve growth factor, and trypsin with estimated Mr of 120,000, 120,000, and 110,000, respectively. PN II was metabolically labeled with [35S]methionine and purified; the metabolically labeled protein formed complexes with EGF-BP. Complexes between purified PN II and EGF-BP bound to human fibroblasts. These results show that the purified protein possesses the properties previously attributed to PN II in cell culture medium.     

Differential expression and stability of foreign genes introduced into human fibroblasts by nuclear versus cytoplasmic microinjection.

We have compared cytoplasmic- and nuclear-delivered, glass needle-mediated microinjection protocols for their ability to support both transient and stable phenotypic expression of reporter gene constructs in non-immortalized human skin fibroblasts cultures. Microinjection of form I (covalently closed circular, supercoiled) plasmid pMC38 DNA into the nucleus of human cells resulted in high levels of transiently expressed p110gag-myc oncoprotein as detected by immunofluorescence microscopy. Likewise, the nuclear delivery of a plasmid construct bearing the entire simian virus 40 genome induced the formation of morphologically transformed foci in approximately 6% of the recipient cell population. In contrast, the introduction of plasmid DNA by the cytoplasmic route proved virtually incapable of supporting either transient gene expression or morphological transformation. In situ autoradiography of cells injected with 3H-labelled plasmid DNA revealed that whereas the material delivered directly into the nucleus was retained by this subcellular compartment for prolonged times (greater than or equal to 48 h), the radiolabelled DNA molecules introduced via the cytoplasmic route did not reach the nucleus and appeared to be substantially degraded within 8 h following injection. These results indicate unequivocally that nuclear injection is the route of choice when monitoring foreign gene expression in human cells.     

Protein unfolding and degradation by the AAA+ Lon protease

AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases.     

Membrane permeabilization by Islet Amyloid Polypeptide

Membrane permeabilization by Islet Amyloid Polypeptide (IAPP) is suggested to be the main mechanism for IAPP-induced cytotoxicity and death of insulin-producing β-cells in type 2 diabetes mellitus (T2DM). The insoluble fibrillar IAPP deposits (amyloid) present in the pancreas of most T2DM patients are not the primary suspects responsible for permeabilization of β-cell membranes. Instead, soluble IAPP oligomers are thought to be cytotoxic by forming membrane channels or by inducing bilayer disorder. In addition, the elongation of IAPP fibrils at the membrane, but not the fibrils themselves, could cause membrane disruption. Recent reports substantiate the formation of an α-helical, membrane-bound IAPP monomer as possible intermediate on the aggregation pathway. Here, the structures and membrane interactions of various IAPP species will be reviewed, and the proposed hypotheses for IAPP-induced membrane permeabilization and cytotoxicity will be discussed.     

Tunicamycin treated fibroblasts secrete a cathepsin B-like protease

We have investigated the effect of tunicamycin on the localization of lysosomal hydrolases in chicken embryo fibroblast cultures. We showed that treatment with tunicamycin (0.05 μg/ml) resulted in a 7–10 fold increase in the cathepsin B-like activity in the culture medium compared to untreated cultures. The protease activity was identified as cathepsin B-like based on 1) substrate specificity (benzoylpro-phe-arg[¹⁴C]anilide is rapidly hydrolyzed), 2) the pH optimum for activity of 5.5, 3) inhibition by thiol reactive compounds, 4) inhibition of the activity by leupeptin but not by pepstatin or phenylmethylsulfonyl fluoride, and 5) by the demonstration of a protease with similar properties in the lysosomal fraction of untreated cultures. The secretion of the cathepsin B-like protease was specific and not due to leakage from damaged cells.     

Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin

When the plasminogen activator urokinase was radioiodinated and incubated at 40 ng/ml in medium conditioned by human foreskin (HF) cells, within 30 min over 80% of the added plasminogen activator was complexed to cell-released protease nexin (PN). The urokinase complexed to PN had little if any activity. Incubation of purified PN with urokinase confirmed that PN is an inhibitor of this plasminogen activator. However, a widely used plasminogen-dependent fibrinolysis assay for plasminogen activator indicated that abundant endogenous plasminogen activator activity co-existed with PN in HF cell-conditioned medium. The source of this activity was electrophoretically and immunologically indistinguishable from urokinase. Furthermore, gel exclusion chromatography showed that about 90% of the urokinase antigen detected in conditioned medium had a molecular weight similar to that of free active urokinase. These paradoxical findings are resolved by evidence that this "PN-resistant urokinase-like" plasminogen activator is actually urokinase proenzyme that is activated by plasmin or conditions in the fibrinolysis assay for plasminogen activator. It is shown that the activated form of HF cell plasminogen activator is sensitive to inhibition by PN. PN may thus be an important component in the cellular regulation of endogenous plasminogen activator activity.     

Serine protease inhibitors modulate chemotactic cytokine production by human lung fibroblasts in vitro

Chemotactic chemokines can be released from lung fibroblasts in response to interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. An imbalance between proteases and antiproteases has been observed at inflammatory sites, and, therefore, protease inhibitors might modulate fibroblast release of chemotactic cytokines. To test this hypothesis, serine protease inhibitors (FK-706, alpha(1)-antitrypsin, or N(alpha)-p-tosyl-L-lysine chloromethyl ketone) were evaluated for their capacity to attenuate the release of neutrophil chemotactic activity (NCA) or monocyte chemotactic activity (MCA) from human fetal lung fibroblasts (HFL-1). Similarly, the release of the chemoattractants IL-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, macrophage colony-stimulating factor, and granulocyte/macrophage colony-stimulating factor, from HFL-1, were evaluated in response to IL-1beta and TNF-alpha. NCA, MCA, and chemotactic cytokines were attenuated by FK-706. However, matrix metalloproteinase inhibitors were without effect, and cysteine protease inhibitors only slightly attenuated chemotactic or cytokine release. These data suggest that IL-1beta and TNF-alpha may stimulate lung fibroblasts to release NCA and MCA by a protease-dependent mechanism and that serine protease inhibitors may attenuate the release.     

Plant transformation by microinjection techniques

Several techniques have been developed for introducing cloned genes into plant cells. Vectorless delivery systems such as PEG-mediated direct DNA uptake (e.g. Pasz-kowski et al. 1984), electroporation (e.g. Shillito et al. 1985), and fusion of protoplasts with liposomes (Deshayes et al. 1985) are routinely used in many experiments (see several chapters of this issue). A wide range of plant species, dicotyledonous as well as monocotyledonous, has been transformed by these vectorless DNA transfer systems. However, the availability of an efficient protoplast regeneration system is a prerequisite for the application of these techniques. For cells with intact cell walls and tissue explants the biological delivery system of virulent Agrobacterium species has been routinely used (for review see Fraley et al. 1986). However, the host range of Agrobacterium restricts the plant species, which can be transformed using this vector system. In addition, all these methods depend on selection systems for recovery of transformants. Therefore a selection system has to be established first for plant species to be transformed. The microinjection technique is a direct physical approach, and therefore host-range independent, for introducing substances under microscopical control into defined cells without damaging them. These two facts differentiate this technique from other physical approaches, such as biolistic transformation and macroinjection (see chapters in this issue). In these other techniques, damaging of cells and random manipulation of cells without optical control cannot be avoided so far. In recent years microinjection technology found its application in plant sciences, whereas this technique has earlier been well established for transformation of animal tissue culture cells (Capecchi 1980) and the production of transgenic animals (Brin-ster et al. 1981, Rusconi and Schaffner 1981). Furthermore, different parameters affecting the DNA transfer via microinjection, such as the nature of microinjected DNA, and cell cycle stage, etc, have been investigated extensively in animal cells (Folger et al. 1982, Wong and Capecchi 1985), while analogous experiments on plant cells are still lacking.     

Mitochondrial membrane permeabilization by HIV-1 Vpr

The mitochondrion is a privileged target for apoptosis-modulatory proteins of viral origin. Thus, viral protein R (Vpr) can target mitochondria and induce apoptosis via a specific interaction with the permeability transition pore complex (PTPC). Vpr cooperates with the adenine nucleotide translocator (ANT) to form large conductance channels and to trigger all the hallmarks of mitochondrial membrane permeabilization (MMP). The Vpr/ANT interaction is direct, since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT, ADP, ATP, or by Bcl-2. Accordingly, Vpr modulates MMP through direct structural and functional interactions with PTPC proteins.     

Tuning permeabilization of microbial cells by three-phase partitioning

Three-phase partitioning of cells was carried out by mixing t-butanol and ammonium sulfate with aqueous suspension of cells. Permeabilized cells formed the interface between aqueous and alcohol layers. A preincubation step in which cells were exposed to just t-butanol was found to tune the selectivity of permeabilized cells of Thermus thermophilus,Saccharomyces cerevisiae, and Escherichia coli. Smaller proteins (green fluorescent protein and lipase with molecular weights of 29 and 34 kDa, respectively) were released with preincubation of 15 min, and penicillin G acylase (∼85 kDa) was released with preincubation of 30 min. The high-molecular-weight proteins (alcohol dehydrogenase from S. cerevisiae and T. thermophilus with molecular weights of 150 and 170 kDa, respectively) were retained even after preincubation of 60 min. The specific activities and electrophoretic analysis of some of the proteins obtained reflected their high purity.     

'Detergent-like' permeabilization of anionic lipid vesicles by melittin

Melittin (MLT), the 26-residue toxic peptide from the European honeybee Apis mellifera, is widely used for studying the principles of membrane permeabilization by antimicrobial and other host-defense peptides. A striking property of MLT is that its ability to permeabilize zwitterionic phospholipid vesicles is dramatically reduced upon the addition of anionic lipids. Because the mechanism of permeabilization may be fundamentally different for the two types of lipids, we examined MLT-induced release of entrapped fluorescent dextran markers of two different molecular masses (4 and 50 kDa) from anionic palmitoyloleoylphosphatidylglycerol (POPG) vesicles. Unlike release from palmitoyloleoylphosphatidylcholine (POPC) vesicles, which is highly selective for the 4 kDa marker, implying release through pores of about 25 A diameter [Ladokhin et al., Biophys. J. 72 (1997) 1762], release from POPG vesicles was found to be non-selective, i.e., 'detergent-like'. Oriented circular dichroism measurements of MLT in oriented POPG and POPC multilayers disclosed that alpha-helical MLT can be induced to adopt a transbilayer orientation in POPC multilayers, but not in POPG multilayers. The apparent inhibition of MLT permeabilization by anionic membranes may thus be due to suppression of translocation ability.