Helicobacter mustelae and Helicobacter pylori bind to common lipid receptors in vitro

Helicobacter pylori is a recently recognized human pathogen causing chronic-active gastritis in association with duodenal ulcers and gastric cancer. Helicobacter mustelae is a closely related bacterium with similar biochemical and morphologic characteristics. H. mustelae infection of antral and fundic mucosa in adult ferrets causes chronic gastritis. An essential virulence property of both Helicobacter species is bacterial adhesion to mucosal surfaces. The aim of this study was to determine whether H. mustelae binds to the same lipids shown previously to be receptors for H. pylori adhesion in vitro. By using thin-layer chromatography overlay and a receptor-based enzyme-linked immunosorbent assay, H. mustelae was found to bind the same receptor lipids as H. pylori, namely, phosphatidylethanolamine and gangliotetraosylceramide. In addition, both H. pylori and H. mustelae bound to a deacylplasmalogen phosphatidylethanolamine. In contrast to H. pylori, H. mustelae binding to receptors was unaffected by motility or viability. Murine monoclonal and bovine polyclonal antibodies against exoenzyme S, and exoenzyme S itself (from Pseudomonas aeruginosa), inhibited binding of H. mustelae to phosphatidylethanolamine and gangliotetraosylceramide. These findings show that H. mustelae binds in vitro to the same lipid receptors as H. pylori and suggest that the adhesion of H. mustelae to such species is mediated by preformed, surface-exposed adhesins which include an exoenzyme S-like protein.     

Identification of a putative target for Rho as the serine-threonine kinase protein kinase N

Rho, a Ras-like small guanosine triphosphatase, has been implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid (LPA) to form stress fibers and focal contacts. The form of RhoA bound to guanosine triphosphate directly bound to and activated a serine-threonine kinase, protein kinase N (PKN). Activated RhoA formed a complex with PKN and activated it in COS-7 cells. PKN was phosphorylated in Swiss 3T3 cells stimulated with LPA, and this phosphorylation was blocked by treatment of cells with botulinum C3 exoenzyme. Activation of Rho may be linked directly to a serine-threonine kinase pathway.     

Characterization of a chromosomal gene encoding type B beta-lactamase in phage group II isolates of Staphylococcus aureus

In contrast to most Staphylococcus aureus isolates in which the gene for staphylococcal beta-lactamase (blaZ) is plasmid borne, isolates typeable by group II bacteriophages frequently carry blaZ on the chromosome. Furthermore, the chromosomal gene encodes the type B variant of staphylococcal beta-lactamase for which the nucleotide and deduced amino acid sequences have not yet been reported. To better understand beta-lactamase production among phage group II staphylococci and the nature of the type B beta-lactamase, we determined the type and amount of enzyme produced by 24 phage group II isolates. Of these isolates, 1 did not produce beta-lactamase, 8 produced the type B enzyme, and 15 produced the type C enzyme. In all eight type B beta-lactamase-producing isolates, blaZ was located on the chromosome. This was in contrast to the type C beta-lactamase-producing isolates, in which blaZ was located on a 21-kb plasmid. The nucleotide sequence corresponding to the leader peptide and the N-terminal 85% of the mature exoenzyme form of type B S. aureus was determined. The deduced amino acid sequence revealed 3 residues in the leader peptide and 12 residues in the exoenzyme portion of the beta-lactamase that differ from the prototypic type A beta-lactamase sequence. These include the serine-to-asparagine change at residue 216 found in the kinetically similar type C enzyme, a threonine-to-lysine change at residue 128 close to the SDN loop (residues 130 to 132), and several substitutions not found in any of the other staphylococcal beta-lactamases. In summary, modern isolates of S. aureus typeable by group II phages produce type B or type C staphylococcal beta-lactamase. The type B gene resides on the chromosome and has a sequence that, when compared to the sequences of the other staphylococcal beta-lactamases, corresponds well with its kinetic properties.     

Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti

Rhizobium meliloti Rm1021 must be able to synthesize succinoglycan in order to invade successfully the nodules which it elicits on alfalfa and to establish an effective nitrogen-fixing symbiosis. Using R. meliloti cells that express green fluorescent protein (GFP), we have examined the nature of the symbiotic deficiency of exo mutants that are defective or altered in succinoglycan production. Our observations indicate that an exoY mutant, which does not produce succinoglycan, is symbiotically defective because it cannot initiate the formation of infection threads. An exoZ mutant, which produces succinoglycan without the acetyl modification, forms nitrogen-fixing nodules on plants, but it exhibits a reduced efficiency in the initiation and elongation of infection threads. An exoH mutant, which produces symbiotically nonfunctional high-molecular-weight succinoglycan that lacks the succinyl modification, cannot form extended infection threads. Infection threads initiate at a reduced rate and then abort before they reach the base of the root hairs. Overproduction of succinoglycan by the exoS96::Tn5 mutant does not reduce the efficiency of infection thread initiation and elongation, but it does significantly reduce the ability of this mutant to colonize the curled root hairs, which is the first step of the invasion process. The exoR95::Tn5 mutant, which overproduces succinoglycan to an even greater extent than the exoS96::Tn5 mutant, has completely lost its ability to colonize the curled root hairs. These new observations lead us to propose that succinoglycan is required for both the initiation and elongation of infection threads during nodule invasion and that excess production of succinoglycan interferes with the ability of the rhizobia to colonize curled root hairs.     

Activation of cyclin-dependent kinase 2 (Cdk2) in growth-stimulated rat astrocytes. Geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E gene expression

The role of the mevalonate cascade in the control of cell cycle progression in astrocytes has been investigated. Serum stimulation of rat astrocytes in primary culture induces the expression of cyclin E followed by the activation of cyclin-dependent kinase 2 (Cdk2) during G1/S transition. The expression of p27, cyclin D1, and the activities of Cdk4 and Cdk-activating kinase (CAK), composed of Cdk7 and cyclin H, were not affected. Serum did, however, stimulate the expression of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase mRNA at mid-G1 phase. Moreover, an inhibitor of HMG-CoA reductase, pravastatin, reduced cyclin E expression and Cdk2 activation and caused G1 arrest in the astrocytes. In contrast, mevalonate and its metabolite, geranylgeranylpyrophosphate (GGPP) but not farnesylpyrophosphate (FPP), reversed the inhibitory effects of pravastatin on cyclin E expression and Cdk2 activation and allowed G1/S transition. Rho small GTPase(s) were geranylgeranylated and translocated to membranes in the presence of GGPP during G1/S transition. The effect of GGPP on cyclin E expression was abolished by botulinum C3 exoenzyme, which specifically inactivates Rho. These data indicate that geranylgeranylated Rho small GTPase(s) are essential for the induction of cyclin E expression, Cdk2 activation, and G1/S transition in rat astrocytes.     

Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells

Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin. Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C. CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics. Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation. Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT. In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization. Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3.     

Lovastatin modulates in vivo and in vitro the plasminogen activator/plasmin system of rat proximal tubular cells: role of geranylgeranylation and Rho proteins

Interstitial fibrosis is one of the most deleterious events during the progression of renal deterioration after renal mass reduction. In vivo, hydroxymethylglutaryl CoA reductase inhibitors (HRI) were shown to reduce progression of glomerulosclerosis, but the mechanisms are still unclear. The present study investigates, in vivo, whether lovastatin, a potent HRI, was able to modulate the plasminogen-plasmin pathway, one of the most efficient systems involved in extracellular matrix remodeling, and characterizes in vitro the cellular mechanisms of these effects. Proximal tubules freshly isolated from rats treated for 2 d with lovastatin (4 mg/kg per d) showed increased tissue-type plasminogen activator (tPA) and urokinase (uPA) activities and antigens. Incubation with lovastatin (5 microM) of proximal tubules isolated from untreated rats induced an increase in tPA and uPA and a decrease in plasminogen activator inhibitor-1 (PAI-1) activities. In vitro, supernatants, cytosols, and membranes of renal proximal tubular cells in primary cultures had no detectable uPA activity, and lovastatin (0.1 to 10 microM) induced an increase in tPA and a decrease in PAI-1 activities and antigens. These effects were reversed by mevalonate and geranylgeranyl-pyrophosphate (GGPP) but not by farnesyl-pyrophosphate or LDL cholesterol. C3 exoenzyme, an inhibitor of the geranylgeranylated-activated Rho protein, reproduced the effect of lovastatin on tPA and PAI- activity and blocked its reversion by GGPP. The effect of lovastatin was associated with a disruption of cellular actin stress fibers, which was reversed by GGPP and reproduced by C3 exoenzyme. In conclusion, HRI can modify the fibrinolytic potential of proximal tubules, most likely via inhibition of geranylgeranylated Rho protein and disruption of the cytoskeleton. The resulting increase of proteolytic activity of tubular cells may serve to prevent extracellular matrix deposition and renal interstitial fibrosis.     

Lysophosphatidic acid-induced association of SHP-2 with SHPS-1: roles of RHO, FAK, and a SRC family kinase

SHPS-1 is an approximately 120 kDa glycosylated receptor like protein that contains three immunoglobulin-like domains in its extracellular region as well as four potential tyrosine phosphorylation and SRC homology 2 (SH2) domain binding sites in its cytoplasmic region. Lysophosphatidic acid (LPA) stimulated the rapid tyrosine phosphorylation of SHPS-1 and its subsequent association with SHP-2, a protein tyrosine phosphatase containing SH2 domains in Rat-1 fibroblasts. LAP-induced tyrosine phosphorylation of SHPS-1 was inhibited by Clostridium botulinum C3 exoenzyme (which inactivates RHO) but not by pertussis toxin. The protein kinase C activator phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) also stimulated tyrosine phosphorylation of SHPS-1; however, down-regulation of protein kinase C by prolonged exposure of cells to TPA did not affect LAP-induced tyrosine phosphorylation of SHPS-1. LPA-induced tyrosine phosphorylation of SHPS-1 was markedly reduced in either focal adhesion kinase (FAK)-deficient mouse cells or CHO cells overexpressing the tyrosine kinase CSK. Overexpression of a catalytically inactivate SHP-2 markedly inhibited MAP kinase activation in response to low concentrations of LPA in CHO cells, whereas overexpression of a wild-type SHPS-1 did enhance this effect of LPA. Furthermore, MAP kinase activation in response to a low concentration of LPA was inhibited by botulinum C3 exoenzyme. These results indicate that LPA-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2 may be mediated by a RHO-dependent pathway that includes FAK and a SRC family kinase. Thus, in addition to its role in receptor tyrosine kinase-mediated MAP kinase activation, the formation of a complex between SHPS-1 and SHP-2 may, in part, play an important role in the activation of MAP kinase in response to low concentrations of LPA.     

The low molecular weight GTPase Rho regulates myofibril formation and organization in neonatal rat ventricular myocytes. Involvement of Rho kinase

The assembly of contractile proteins into organized sarcomeric units is one of the most distinctive features of cardiac myocyte hypertrophy. In a well characterized in vitro model system using cultured neonatal rat ventricular myocytes, a subset of G protein-coupled receptor agonists has been shown to induce actin-myosin filament organization. Pretreatment of myocytes with C3 exoenzyme ADP-ribosylated Rho and inhibited the characteristic alpha1-adrenergic receptor agonist-induced myofibrillar organization, suggesting involvement of the Rho GTPase in cardiac myofibrillogenesis. We used adenoviral mediated gene transfer to examine the effects of activated Rho and inhibitory mutants of one of its effectors, Rho kinase, in myocytes. Rho immunoreactivity was increased in the particulate fraction of myocytes infected with a recombinant adenovirus expressing constitutively activated Rho. Rho-infected cells demonstrated a striking increase in the assembly and organization of sarcomeric units and in the expression of the atrial natriuretic factor protein. These Rho-induced responses were markedly inhibited by co-infection with adenoviruses expressing putative dominant negative forms of Rho kinase. A parallel pathway involving Ras-induced myofibrillar organization and atrial natriuretic factor expression was only minimally affected. alpha1-Adrenergic receptor agonist-induced myofibrillogenesis was inhibited by some but not all of the Rho kinase mutants. Our data demonstrate that activated Rho has profound effects on myofibrillar organization in cardiac myocytes and suggest that Rho kinase mediates Rho-induced hypertrophic responses.     

Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells

Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity. In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates and inactivates Rho proteins. Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists. Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial. GTPgammaS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment. Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPgammaS on PLD. ATP produced a significant enhancement of the in vitro effect of GTPgammaS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases. However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP2.     

Gbeta gamma-independent coupling of alpha2-adrenergic receptor to p21(rhoA) in preadipocytes

In preadipocytes, alpha2-adrenergic receptor (alpha2-AR) stimulation leads to a Gi/Go-dependent rearrangement of actin cytoskeleton. This is characterized by a rapid cell spreading, the formation of actin stress fibers, and the increase in tyrosyl phosphorylation of the focal adhesion kinase (pp125(FAK)). These cellular events being tightly controlled by the small GTPase p21(rhoA), the existence of a Gi/Go-dependent coupling of alpha2-AR to p21(rhoA) in preadipocytes was proposed. In alpha2AF2 preadipocytes (a cell clone derived from the 3T3F442A preadipose cell line and which stably expresses the human alpha2C10-adrenergic receptor) alpha2-adrenergic-dependent induction of cell spreading, formation of actin stress fibers, and increase in tyrosyl phosphorylation of pp125(FAK) were abolished by pretreatment of the preadipocytes with the C3 exoenzyme, a toxin which impairs p21(rhoA) activity by ADP-ribosylation. Conversely, C3 exoenzyme had no effect on the alpha2-adrenergic-dependent increase in tyrosyl phosphorylation and shift of ERK2 mitogen-activated protein kinase. alpha2-Adrenergic stimulation also led to an increase in GDP/GTP exchange on p21(rhoA), as well as to an increase in the amount of p21(rhoA) in the particulate fraction of alpha2AF2 preadipocytes. Stable transfection of alpha2AF2 preadipocytes with the COOH-terminal domain of betaARK1 (betaARK-CT) (a blocker of Gbeta gamma-action), strongly inhibited the alpha2-adrenergic-dependent increase in tyrosyl phos- phorylation and shift of ERK2, without modification of the tyrosyl phosphorylation of pp125(FAK) and spreading of preadipocytes. These results show that alpha2-adrenergic-dependent reorganization of actin cytoskeleton requires the activation of p21(rhoA) in preadipocytes. Conversely to the activation of the p21(ras)/mitogen-activated protein kinase pathway, the alpha2-adrenergic activation of p21(rhoA)-dependent pathways are independent of the beta gamma-subunits of heterotrimeric G proteins.     

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.     

rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells

Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic. Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin. We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein. The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase. Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA. Attachment of S. flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase. Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.     

Inhibition of receptor signaling to phospholipase D by Clostridium difficile toxin B. Role of Rho proteins

Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5'-O-(thio)triphosphate (GTP gamma S) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF4- and GTP gamma S, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTP gamma S-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.     

Signaling pathways involved in thrombin-induced cell protection

This study examined the signal transduction pathways involved in thrombin-induced neuroprotection and compares these results with those of a similar study of thrombin-induced neuronal death. In thrombin-induced protection of astrocytes from hypoglycemia, pretreatment of astrocytes with tyrosine or serine/threonine kinase inhibitors, cytochalasin D, or exoenzyme C3, a potent inhibitor of the small GTPase RhoA, attenuated thrombin-induced protection. These same inhibitors were previously shown to block thrombin-induced cell death, implying a similarity in the cell death and cell-protective pathways. Biochemical assays determined that thrombin increased available RhoA activity, although more slowly and to a lesser extent than occurs in thrombin-induced cell death. A clear difference in these pathways was revealed when a time course study of thrombin-induced cell death indicated that unlike thrombin-induced protection, cells must be exposed to thrombin for >16 h to irreversibly enter the cell death pathway. Addition of lower doses of thrombin every 24 h also induced cell death. These studies indicate that exposure of cells to micromolar concentrations of thrombin alone does not induce cell death, but the continued exposure to thrombin is required. Thus the cell death and protective pathways may share initial signaling proteins, but differences in the amplitude as well as the duration of the signal may result in different final pathways.     

Lipoprotein nature of Bacillus licheniformis membrane penicillinase

Membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis bears a striking resemblance to the major outer membrane lipoprotein of Escherichia coli. It can be specifically labeled in vivo with [3H]glycerol, [35S]cysteine, or [3H]palmitate but not by [32P]orthophosphate. The labeled residues are located at or near the NH2 terminus of the membrane penicillinase because they can be completely removed by trypsin which cleaves a hydrophobic peptide(s) from the NH2 terminus, thereby rendering the enzyme hydrophilic. The membrane penicillinase produced by the 749/C gene carried in E. coli on phage lambda is similar to the enzyme formed in strain 749/C itself. The peptide antibiotic globomycin, which prevents processing of the E. coli prolipoprotein, severely inhibited the attachment of [3H]palmitate or [3H]glycerol to the 749/C enzyme (either in B. licheniformis 749/C or in E. coli), blocked the accumulation of penicillinase in the plasma membrane, and enhanced the formation of exoenzyme. Under the same conditions, globomycin does not prevent the attachment of palmitate or glycerol to the E. coli prolipoprotein but inhibits processing of the modified precursor to the mature lipoprotein. These results are in contrast with the lack of effect of globomycin on the RTEM-beta-lactamase of E. coli which has no detectable hydrophobic membrane form and was not labeled with palmitate or glycerol.     

Regulation of alphaIIb beta3 function in human B lymphocytes

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.     

Career planning and development

Germinating conidiospores (conidia) of Aspergillus nidulans amino acid-requiring strains are hypersensitive to heat, oxidative stress, UV radiation and chemical mutagens when compared with other strains. They also showed an increased mutation rate. Sensitivity to stress conditions has been correlated with an abnormal RAS/cAMP pathway in mutants of S. cerevisiae. We suggest that the RAS/cAMP pathway is defective in germinating conidia of Aspergillus amino acid auxotrophs and that this is responsible for suppressing DNA repair and conferring sensitivity to oxidative stress and heat shock.