Secretion, purification, and characterisation of barley alpha-amylase produced by heterologous gene expression in Aspergillus niger

The role of the actin cytoskeleton and/or GTPases of the Rho/Rac-family in glucose transport regulation was investigated in 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho/Rac (Clostridium difficile toxin B and Clostiridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake rates in 3T3-L1 fibroblasts. In parallel, the toxins produced morphological alterations of the cells reflecting disruption of the actin cytoskeleton. Both toxins reduced the maximum response to insulin but failed to alter the half-maximally stimulating concentrations of insulin. In 3T3-L1 adipocytes, the lethal toxin reduced the effect of insulin on 2-DOG uptake, whereas toxin B and C2 failed to affect glucose transport or cell morphology. When cells were exposed to the toxins after treatment with insulin, both toxin B and the lethal toxin, in contrast to the phosphatidylinositol (PI) 3-kinase inhibitor wortmannin, failed to reduce the 2-DOG uptake rates. Thus, both translocation to the plasma membrane and internalization of glucose transporters were inhibited by the toxins, whereas the PI 3-kinase inhibitor selectively affects translocation. The data suggest that the effects of the clostridial toxins on trafficking of glucose transporters are mediated by the depolymerization of the actin cytoskeleton and are an indirect consequence of Rho or Rac inactivation. It is suggested that pathways signalling through Rac or Rho may play a modulatory role in glucose transport regulation through their effects on the actin network.     

Bradycardia induced by intravascular versus direct stimulation of the vagus nerve

Ricin is a heterodimeric cytotoxin composed of RTB, a galactose binding lectin, and RTA, an enzymatic N-glycosidase. The toxin is endocytosed, and after intracellular routing, RTA is translocated to the cytoplasm where it inactivates ribosomes resulting in a loss of host cell protein synthesis and cell death. We show for the first time that the cytotoxicity against cultured T cells by several RTA mutants is directly proportional to the enzyme activity of RTA, suggesting this is a reliable system to measure translocation effects. Large discrepancies between cytotoxicity and enzyme action for a given pair of toxins are therefore attributable to differences in cell binding, uptake, or membrane translocation. Fluid phase uptake and cytotoxicity of isolated RTA are essentially identical to that of the single chain toxin PAP. This important finding suggests that RTA, and the A chain of class 2 RIPs in general, has not evolved special translocation signals to complement the increased target cell binding facilitated by RTB. Experiments with the lectin RCA and with ebulin suggest those toxins have diminished cytotoxicity probably mediated by comparative deficiencies in B chain binding. Addition of a KDEL sequence to RTA increases fluid phase uptake, consistent with the notion that transport to the ER is important for cytotoxicity. Fusion of MBP or GST to the amino terminus of RTA has little effect on enzyme action or cytotoxicity. This result is not altered by protease inhibitors, suggesting the fusion proteins are probably not cleaved prior to translocation of the toxic A chain and implying that the toxins can carry large passenger proteins into the cytoplasm, an observation with interesting potential for analytical and therapeutic chemistry.     

The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.     

The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins

BACKGROUND: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. RESULTS: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP. CONCLUSIONS: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.     

Stepwise transplantation of an active site loop between heat-labile enterotoxins LT-II and LT-I and characterization of the obtained hybrid toxins

Members of the cholera toxin family, including Escherichia coli heat-labile enterotoxins LT-I and LT-II, catalyze the covalent modification of intracellular proteins by transfer of ADP-ribose from NAD to a specific arginine of the target protein. The ADP-ribosylating activity of these toxins is located in the A-subunit, for which LT-I and LT-II share a 63% sequence identity. The flexible loop in LT-I, ranging from residue 47 to 56, closes over the active site cleft. Previous studies have shown that point mutations in this loop have dramatic effects on the activity of LT-I. Yet, in LT-II the sequence of the equivalent loop differs at four positions from LT-I. Therefore five mutants of the active site loop were created by a stepwise replacement of the loop sequence in LT-I with virtually all the corresponding residues in LT-II. Since we discovered that LT-II had no activity versus the artificial substrate diethylamino-benzylidine-aminoguanidine (DEABAG) while LT-I does, our active site mutants most likely probe the NAD binding, not the arginine binding region of the active site. The five hybrid toxins obtained (Q49A, F52N, V53T, Q49V/F52N and Q49V/F52N/V53T) show (i) great differences in holotoxin assembly efficiency; (ii) decreased cytotoxicity in Chinese hamster ovary cells; and (iii) increased in vitro enzymatic activity compared with wild type LT-I. Specifically, the three mutants containing the F52N substitution display a greater Vmax for NAD than wild type LT-I. The enzymatic activity of the V53T mutant is significantly higher than that of wild type LT-I. Apparently this subtle variation at position 53 is beneficial, in contrast to several other substitutions at position 53 which previously had been shown to be deleterious for activity. The most striking result of this study is that the active site loop of LT-I, despite great sensitivity for point mutations, can essentially be replaced by the active site loop of LT-II, yielding an active 'hybrid enzyme' as well as 'hybrid toxin'.     

A role for tissue factor in cell adhesion and migration mediated by interaction with actin-binding protein 280

Tissue factor (TF), the protease receptor initiating the coagulation system, functions in vascular development, angiogenesis, and tumor cell metastasis by poorly defined molecular mechanisms. We demonstrate that immobilized ligands for TF specifically support cell adhesion, migration, spreading, and intracellular signaling, which are not inhibited by RGD peptides. Two-hybrid screening identified actin-binding protein 280 (ABP-280) as ligand for the TF cytoplasmic domain. Extracellular ligation of TF is necessary for ABP-280 binding. ABP-280 recruitment to TF adhesion contacts is associated with reorganization of actin filaments, but cytoskeletal adaptor molecules typically found in integrin-mediated focal contacts are not associated with TF. Chimeric molecules of the TF cytoplasmic domain and an unrelated extracellular domain support cell spreading and migration, demonstrating that the extracellular domain of TF is not involved in the recruitment of accessory molecules that influence adhesive functions. Replacement of TF's cytoplasmic Ser residues with Asp to mimic phosphorylation enhances the interaction with ABP-280, whereas Ala mutations abolish coprecipitation of ABP-280 with immobilized TF cytoplasmic domain, and severely reduce cell spreading. The specific interaction of the TF cytoplasmic domain with ABP-280 provides a molecular pathway by which TF supports tumor cell metastasis and vascular remodeling.     

Isoform specificity in the relationship of actin to dendritic spines

Dendritic spines contain high concentrations of actin, but neither the isoforms involved nor the mechanism of accumulation is known. In situ hybridization with specific probes established that beta- and gamma-cytoplasmic actins are selectively expressed at high levels by spine-bearing neurons. Transfecting cultured hippocampal neurons with epitope-tagged actin isoforms showed that cytoplasmic beta- and gamma-cytoplasmic actins are correctly targeted to spines, whereas alpha-cardiac muscle actin, which is normally absent from neurons, formed aggregates in dendrites. The transfected actin cDNAs contained only coding domains, suggesting that spine targeting involves amino acid sequences in the proteins, an interpretation supported by experiments with chimeric cDNAs in which C-terminal actin sequences were found to be determinative in spine targeting. By contrast to actin, microtubule components, including tubulin and MAP2, were restricted to the dendritic shaft domain. The close association of cytoplasmic actins with spines together with their general involvement in cell surface motility further supports the idea that actin motility-based changes in spine shape may contribute to synaptic plasticity.     

GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels

GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine-activated potassium current (IKACh) channel in pacemaker cells of the heart. The channel is activated by direct binding of G-protein Gbetagamma subunits. The GIRK1 subunit is atypical in the GIRK family in having a unique ( approximately 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK family member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagged GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surface localization. Furthermore, GIRK1 homomultimers reside in core-glycosylated and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [35S]Methionine pulse-labeling experiments demonstrated that GIRK4 associates with GIRK1 either during or shortly after subunit synthesis. Mutant and chimeric channel subunits were utilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta374-501 resulted in an intracellular GIRK1 subunit that produced normal IKACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis of the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appeared intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in the processing and cell surface localization of IKACh in vivo.     

A new monoclonal antibody, mAb 4A12, identifies a role for the glycosaminoglycan (GAG) binding domain of RANTES in the antiviral effect against HIV-1 and intracellular Ca2+ signaling

The beta-chemokine RANTES (regulated on activation, normal T cell expressed and secreted) suppresses the infection of susceptible host cells by macrophage tropic strains of HIV-1. This effect is attributed to interactions of this chemokine with a 7-transmembrane domain receptor, CCR5, that is required for virus-cell fusion and entry. Here we identify domains of RANTES that contribute to its biological activities through structure-function studies using a new monoclonal antibody, mAb 4A12, isolated from mice immunized with recombinant human RANTES. This monoclonal antibody (mAb) blocked the antiviral activity of RANTES in infectivity assays with HIV-1Bal, and inhibited the mobilization of intracellular Ca2+ elicited by RANTES, yet recognized this chemokine bound to cell surfaces. Epitope mapping using limited proteolysis, reversed phase high-performance liquid chromatography, and mass spectrometry suggest that residues 55-66 of RANTES, which include the COOH-terminal alpha-helical region implicated as the glycosaminoglycan (GAG) binding domain, overlap the determinant recognized by mAb 4A12. This is supported by affinity chromatography studies, which showed that RANTES could be eluted specifically by heparin from a mAb 4A12 immunoaffinity matrix. Removal of cell surface GAGs by enzymatic digestion greatly reduced the ability of mAb 4A12 to detect RANTES passively bound on cell surfaces and abrogated the ability of RANTES to elicit an intracellular Ca2+ signal. Taken together, these studies demonstrate that the COOH-terminal alpha-helical region of RANTES plays a key role in GAG-binding, antiviral activity, and intracellular Ca2+ signaling and support a model in which GAGs play a key role in the biological activities of this chemokine.     

Modification of Ras in eukaryotic cells by Pseudomonas aeruginosa exoenzyme S

Genetic and functional data suggest that Pseudomonas aeruginosa exoenzyme S (ExoS), an ADP-ribosyltransferase, is translocated into eukaryotic cells by a bacterial type III secretory mechanism activated by contact between bacteria and host cells. Although purified ExoS is not toxic to eukaryotic cells, ExoS-producing bacteria cause reduced proliferation and viability, possibly mediated by bacterially translocated ExoS. To investigate the activity of translocated ExoS, we examined in vivo modification of Ras, a preferred in vitro substrate. The ExoS-producing strain P. aeruginosa 388 and an isogenic mutant strain, 388DeltaexoS, which fails to produce ExoS, were cocultured with HT29 colon carcinoma cells. Ras was found to be ADP-ribosylated during coculture with 388 but not with 388DeltaexoS, and Ras modification by 388 corresponded with reduction in HT29 cell DNA synthesis. Active translocation by bacteria was found to be required, since exogenous ExoS, alone or in the presence of 388DeltaexoS, was unable to modify intracellular Ras. Other ExoS-producing strains caused modification of Ras, indicating that this is not a strain-specific event. ADP-ribosylation of Rap1, an additional Ras family substrate for ExoS in vitro, was not detectable in vivo under conditions sufficient for Ras modification, suggesting possible ExoS substrate preference among Ras-related proteins. These results confirm that intracellular Ras is modified by bacterially translocated ExoS and that the inhibition of target cell proliferation correlates with the efficiency of Ras modification.     

Quantitative analysis of bacterial toxin affinity and specificity for glycolipid receptors by surface plasmon resonance

The primary virulence factors of many pathogenic bacteria are secreted protein toxins which bind to glycolipid receptors on host cell surfaces. The binding specificities of three such toxins for different glycolipids, mainly from the ganglioside series, were determined by surface plasmon resonance (SPR) using a liposome capture method. Unlike microtiter plate and thin layer chromatography overlay assays, the SPR/liposome methodology allows for real time analysis of toxin binding under conditions that mimic the natural cell surface venue of these interactions and without any requirement for labeling of toxin or receptor. Compared to conventional assays, the liposome technique showed more restricted oligosaccharide specificities for toxin binding. Cholera toxin demonstrated an absolute requirement for terminal galactose and internal sialic acid residues (as in GM1) with tolerance for substitution with a second internal sialic acid (as in GD1b). Escherichia coli heat-labile enterotoxin bound to GM1 and tolerated removal or extension of the internal sialic acid residue (as in asialo-GM1 and GD1b, respectively) but not substitution of the terminal galactose of GM1. Tetanus toxin showed a requirement for two internal sialic acid residues as in GD1b. Extension of terminal galactose with a single sialic acid was tolerated to some extent. The SPR analyses also yielded rate and affinity constants which are not attainable by conventional assays. Complex binding profiles were observed in that the association and dissociation rate constants varied with toxin:receptor ratios. The sub-nanomolar affinities of cholera toxin and heat-labile enterotoxin for liposome-anchored gangliosides were attributable largely to very slow dissociation rate constants. The SPR/liposome technology should have general applicability in the study of glycolipid-protein interactions and in the evaluation of reagents designed to interfere with these interactions.     

Recovery of staphylococcal enterotoxin from foods by affinity chromatography

Extraction, concentration, and serological detection of staphylococcal enterotoxins from foods are laborious and time consuming. By exposing food extracts to an insoluble matrix tagged with specific anti-enterotoxin B, we have been able to recover the toxin from foods in a sensitive and rapid way. After mixing the reagents for 2 h at room temperature, immunoglobulin G antibodies were attached to CNBr-activated Sepharose 4B at pH 8.5 (0.1 M carbonate buffer with 0.5 M NaCl). Sepharose-antibody complex (1 ml) specifically recovered 0.1 to 30 mug of enterotoxin B from 400 ml of food extract (100 g of food) after mixing for 2 h at 4 C. The Sepharose-antibody-toxin complex was washed with 0.02 M phosphate-buffered saline at pH 7.2, and the toxin was dissociated by 2 to 4 ml of 0.2 M HCl-glycine plus 0.5 M NaCl buffer at pH 2.8. The recovered enterotoxin was free of interfering food components and could be detected serologically. Work to couple antibodies A, B, C, D, and E to Sepharose to recover all five toxins in one step is under study.     

Biogenesis, cellular localization, and functional activation of the heat-stable enterotoxin receptor (guanylyl cyclase C)

Enterotoxigenic Escherichia coli elaborate a peptide called heat-stable enterotoxin (ST), which binds to and activates the intestinal ST receptor (STaR). STaR, also known as guanylyl cyclase C (GC-C), is a member of the transmembrane guanylyl cyclase receptor family. The mRNA for STaR encodes an approximately 120 kDa protein with the N-terminal ligand binding domain on the cell surface. Ligand affinity cross-linking studies have previously demonstrated several species of potential ST binding proteins, ranging in size from approximately 50 to 160 kDa. Although these smaller forms of STaR (50-80 kDa) have been proposed to act in vivo as toxin binding proteins, their biogenesis and localization have not previously been examined. Using pulse labeling in vivo and synchronized translation in vitro, we demonstrate that these smaller forms represent incomplete translational products and are not formed through limited proteolysis of the full-length receptor, as had previously been believed. We determined, using fluorescence confocal microscopy and surface labeling, that only approximately 25% of cellular receptors are expressed at the surface, while the remaining population is retained within the endoplasmic reticulum. Only full-length receptor is found at the surface of the cell, indicating this to be the biologically active form of STaR responsible for interacting with the heat-stable enterotoxin and other luminal intestinal peptides. The large intracellular receptor population, and potential for function before translocation to the cell surface, may impact on how pharmacologic modulators of this clinically important receptor are designed.     

The Arg7Lys mutant of heat-labile enterotoxin exhibits great flexibility of active site loop 47-56 of the A subunit

The heat-labile enterotoxin from Escherichia coli (LT) is a member of the cholera toxin family. These and other members of the larger class of AB5 bacterial toxins act through catalyzing the ADP-ribosylation of various intracellular targets including Gs alpha. The A subunit is responsible for this covalent modification, while the B pentamer is involved in receptor recognition. We report here the crystal structure of an inactive single-site mutant of LT in which arginine 7 of the A subunit has been replaced by a lysine residue. The final model contains 103 residues for each of the five B subunits, 175 residues for the A1 subunit, and 41 residues for the A2 subunit. In this Arg7Lys structure the active site cleft within the A subunit is wider by approximately 1 A than is seen in the wild-type LT. Furthermore, a loop near the active site consisting of residues 47-56 is disordered in the Arg7Lys structure, even though the new lysine residue at position 7 assumes a position which virtually coincides with that of Arg7 in the wild-type structure. The displacement of residues 47-56 as seen in the mutant structure is proposed to be necessary for allowing NAD access to the active site of the wild-type LT. On the basis of the differences observed between the wild-type and Arg7Lys structures, we propose a model for a coordinated sequence of conformational changes required for full activation of LT upon reduction of disulfide bridge 187-199 and cleavage of the peptide loop between the two cysteines in the A subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of sequences required for the cytotoxic action of a chimeric toxin composed of Pseudomonas exotoxin and transforming growth factor alpha

Chimeric toxins composed of transforming growth factor alpha fused to mutant forms of Pseudomonas exotoxin bind to the EGF receptor and kill cells bearing these receptors. In early experiments, the binding domain of Pseudomonas exotoxin was deleted and replaced with TGF alpha to make TGF alpha-PE40. This chimeric toxin required proteolytic processing within the target cell to be converted to its active form (Siegall et al. (1989) FASEB J. 3, 2647-2652). Subsequently, recombinant toxins that do not require proteolytic processing were constructed by inserting TGF alpha near the carboxyl terminus of domain III and deleting toxin residues up to the processing site at position 280. In addition, the carboxyl terminus of this toxin was converted from REDLK to KDEL to increase its activity. Recombinant toxins of this type, termed PE37/TGF alpha/KDEL, are about 100-fold more potent than TGF alpha-PE40. To determine if other sequences can be removed from such chimeric toxins to make a smaller molecule that can penetrate tissues better, we have carried out a deletion analysis of sequences present within domains II and Ib. We find that all of domain Ib and a portion of domain II can be deleted without significant loss of cytotoxic activity, but larger deletions extending further into domain II lose cytotoxic activity. We also find that inserting a small linking peptide (Gly)4Ser between residual sequences in domain II and domain III, in molecules with diminished cytotoxic activity, enhances cytotoxicity suggesting that one role of domain Ib is to prevent undesirable interactions between domains II and III.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mast cell histamine-induced calcium transients in cultured human peritoneal mesothelial cells

OBJECTIVE: Peritoneal inflammation results from a complex interplay of events initiated by macrophage activity in response to infection, with the stimulation of mesothelial cell cytokine release amplifying the recruitment of blood-borne defense cells to the site of injury. Resident peritoneal mast cells may add to this complexity with mast cell derived cytokines released during this cascade. This study examined the influence of histamine, a mast cell-derived inflammatory mediator, on the initial activation of human peritoneal mesothelial cells (HPMC) by intracellular free calcium (Ca2+(i)) mobilization, and changes to the actin cytoskeleton. DESIGN: HPMC signal transduction was examined in response to histamine (1.0 mmol/L) compared to fetal bovine serum (FBS) (0.1%) and 4-br-A23187 (1.0 micromol/L). Intracellular free calcium was measured in fura-2 loaded cells with and without external calcium (Ca2+(ext)), or Ca2+(ext) with verapamil (100 micromol/L). Following treatment with agonists, HPMC actin cytoskeleton was stained using direct immunocytochemistry. RESULTS: HPMC responded to histamine with a twofold transient rise in Ca2+(i) which returned to the baseline, in contrast with FBS- and A23187-induced Ca2+(i) transients, which returned to elevated resting values. In the absence of Ca2+(ext), all agents produced a calcium transient indicative of calcium release from intracellular stores. Histamine induced calcium-dependent changes to the cytoskeleton and cellular organization, including increased actin stress fibers. CONCLUSION: Histamine produced large specific receptor-mediated calcium transients in HPMC, which included components of calcium release from intracellular stores and receptor-mediated calcium influx processes. The observed response to histamine raises the possibility that histamine derived from resident mast cells may modulate mesothelial cell function, in part by calcium-dependent pathways, and influence the performance of the peritoneal membrane during peritoneal dialysis.     

Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42

We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTPgammaS to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTPgammaS appeared to act via a small G-protein, since it was active in lysates ofD. discoideum mutants missing either the alpha2- or beta-subunit of the heterotrimeric G-protein required for chemoattractant-induced actin polymerization in living cells. Furthermore, recombinant Cdc42, but not Rho or Rac, induced polymerization in the cell-free system. The Cdc42-induced increase in filamentous actin required GTPgammaS binding and was inhibited by a fragment of the enzyme PAK1 that binds Cdc42. In a high speed supernatant, GTPgammaS alone was ineffective, but GTPgammaS-loaded Cdc42 induced actin polymerization, suggesting that the response was limited by guanine nucleotide exchange. Stimulating exchange by chelating magnesium, by adding acidic phospholipids, or by adding the exchange factors Cdc24 or Dbl restored the ability of GTPgammaS to induce polymerization. The stimulation of actin polymerization did not correlate with PIP2 synthesis.     

An EGF-pseudomonas exotoxin A recombinant protein with a deletion in toxin binding domain specifically kills EGF receptor bearing cells

We constructed two chimeric toxins; one composed of epidermal growth factor (EGF) and pseudomonas exotoxin A (PE), designated EGF-PE and the other composed of EGF and PE with a deletion of the Ia domain (cell-binding domain), designated EGF-PE (delta Ia). Both chimeric toxins reacted with anti-EGF and anti-PE antibodies. The cell-killing experiments showed that EGF-PE, but not EGF-PE (delta Ia), was cytotoxic to the murine fibroblast cell line NR6, which carried the PE receptor, but not the EGF receptor. However, after NR6 was transfected with DNA for the expression of human EGF receptor, the transfected cell line, designated NRHER5, over-expressed human EGF receptors and became sensitive to EGF-PE(delta IA). The cytotoxicity of EGF-PE(delta Ia), but not EGF-PE, to NRHER5 can be completely blocked by an excess amount of EGF. To completely reverse the cytotoxicity of EGF-PE on NRHER5, both the EGF receptor pathway and the PE receptor pathway need to be blocked. These results suggest that EGF-PE exhibits both EGF and PE binding activities, while EGF-PE(delta IA) possesses only EGF binding activity. Thus, EGF-PE(delta Ia) may be a better chimeric toxin than EGF-PE in terms of target specificity to EGF receptor bearing cells. We, therefore, examined the cytotoxicity of EGF-PE(delta Ia) to various human cancer cell lines. We find that human cancer cells containing more EGF receptors are more sensitive to EGF-PE(delta Ia).     

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.