Pores in the outer membrane of Escherichia coli K12: involvement of proteins b and e in the functioning of pores for nucleotides.

Summary Mutants lacking outer membrane proteins were studied in order to investigate the role of these proteins in the functioning of aqueous pores through the outer membrane. Protein b is involved in the functioning of pores through which low concentrations of adenosine-monophosphate (AMP), guanosine-monophosphate (GMP), bis(paranitrophenyl)-phosphate (bis-PNPP) and, although less pronounced, also cytidine-monophosphate (CMP) enter the cell. In the absence of the receptor protein of phage T6, which facilitates the permeation of various nucleosides (Hantke, 1976), protein b plays a major role in the uptake of adenosine. Proteins c and d and the receptor proteins of phages lambda and T6 do not have a pore function for AMP, GMP, CMP and bis-PNPP. However, a newly discovered peptidoglycan-associated protein, protein e, can also mediate the permeation of the latter four components, but not of adenosine, through the outer membrane.Protein b does not play a major role in the uptake of higher concentrations of AMP. Obviously mainly other pores are used under those conditions. The results strongly suggest that at low concentrations of nucleoside monophosphates (and possibly of other nutrients) protein b functions as a specific pore.     

Functions related to the receptor protein specified by the tsx gene of Escherichia coli

The receptor protein for the phage T6 and colicin K, coded by the tsx gene, facilitated the diffusion of all nucleosides and deoxynucleosides except cytidine and deoxcytidine through the outer membrane of Escherichia coli K-12 and Escherichia coli B. The tsx protein was coregulated with the nucleoside uptake system. Constitutive cytR and deoR mutants contained higher amounts of this protein than wild type strains. There was a good correlation between the initial rate of nucleoside uptake and the adsorption rate of phage T6. From the observation that nucleosides did not compete with each other in the translocation across the outer membrane and that they did not inhibit T6 adsorption it was concluded that the tsx protein forms a pore to which nucleosides have only little if any binding affinity.A major outer membrane protein specified by the ompA gene influenced the function of the tsx protein. Outer membranes of ompA mutants showed an enhanced binding of colicin K but the strains were colicin K insensitive (tolerant). The T6 phage adsorbed at the same rate and plated with the same efficiency as to ompA ⁺ strains. The uptake rate of thymidine and of adenosine was reduced by 16–33% in ompA mutants.The adsorption rate of phage T6 on mutants with altered lipopolysaccharide was the same or even higher than on wild type strains. However the plating efficiency was reduced ranging from 0–46%. Lipopolysaccharide plays no role in the primary adsorption of phage T6 but it is apparently required in a later step of the infection process.Non Standard Abbreviations LPS lipopolysaccharide - cAMP-CRP complex of cyclic adenosine 3,5-monophosphate (cAMP) and its receptor protein (CRP)     

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.     

Pore-forming activity of the Tsx protein from the outer membrane of Escherichia coli. Demonstration of a nucleoside-specific binding site

The Tsx protein from the outer membrane of Escherichia coli is known to be involved in the permeation of nucleosides across the outer membrane under limiting substrate conditions. We purified Tsx from an E. coli strain that overproduces Tsx. The purified protein was still functional since it could neutralize the Tsx-specific bacteriophage T6 in vitro. When the purified Tsx was reconstituted into a lipid bilayer, there was a large increase of the membrane conductance, indicating pore-forming activity of Tsx in vitro. This increase could be strongly blocked with adenosine and to a much lesser extent with cytidine. Titration of the pore conductance with adenosine or cytidine suggested the presence of a binding site for nucleosides in the Tsx pore, with a Ks of 6 X 10(-4) and 2 X 10(-2) M for adenosine and cytidine, respectively. We propose that the Tsx protein functions in vivo as a pore that specifically facilitates the permeation of nucleosides across the outer membrane due to its binding site for nucleosides.     

Integration of the overproduced bacteriophage T5 receptor protein in the outer membrane of Escherichia coli

The tonA gene codes for an outer membrane protein, a receptor of phage T5, the TonA protein. Strains harboring pLG513, a multicopy plasmid in which the tonA gene has been cloned, overproduced TonA protein, which appeared in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell envelope proteins as a 78,000-molecular-weight protein. Identical results have been observed by Plastow et al. (FEBS Lett. 131:262-264, 1981) with plasmid pLC19-19, in which the tonA gene has also been cloned. The activity of the TonA protein, measured by its capacity to inactivate phage T5, increased by five- to sixfold in purified envelopes of cells harboring pLG513 compared with cells lacking the plasmid. Solubilization of the cytoplasmic membrane by Triton-Mg2+ treatment did not increase this activity. However, partial solubilization of outer membrane proteins by Triton-EDTA unmasked further T5 receptor activity, resulting in a final increase of around 50-fold, a value more consistent with the expected gene dosage effect. Treatment of whole cells by trypsin in conditions in which trypsin is allowed to enter the outer membrane revealed that part of the overproduced T5 receptors were embedded in the outer membrane and masked by a trypsin-sensitive protein. In addition, no T5 receptor activity was detected in either the periplasmic space or the cytoplasm. These results suggest that all of the overproduced TonA molecules were synthesized in an active form and integrated in the outer membrane, but only a small fraction could be reached or recognized by phage T5 in vivo.     

Spatiotemporal control of cyclic AMP immunomodulation through the PKA-Csk inhibitory pathway is achieved by anchoring to an Ezrin-EBP50-PAG scaffold in effector T cells

A variety of immunoregulatory signals to effector T cells from monocytes, macrophages and regulatory T cells act through cyclic adenosine monophosphate. In the effector T cell, the protein kinase A (PKA) type I isoenzyme localizes to lipid rafts during T cell activation and modulates directly the proximal events that take place after engagement of the T cell receptor. The most proximal target for PKA phosphorylation is C-terminal Src kinase (Csk), which initiates a negative signal pathway that fine-tunes the T cell activation process. The A kinase anchoring protein Ezrin colocalizes PKA and Csk by forming a supramolecular signaling complex consisting of PKA, Ezrin, Ezrin/radixin/moesin (ERM) binding protein of 50 kDa (EBP50), phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (GEMs) (PAG) and Csk.     

Adenosine Inhibits Histamine-Induced Phosphoinositide Hydrolysis Mediated via Pertussis Toxin-Sensitive G Protein in Human Astrocytoma Cells

The effect of adenosine on phosphoinositide hydrolysis was examined in 1321N1 human astrocytoma cells. Adenosine, L-N6-phenylisopropyladenosine (L-PIA), and 5'-(N-ethylcarboxamido)adenosine (NECA) inhibited histamine-stimulated accumulation of inositol phosphates in a concentration-dependent manner. The potency order of adenosine analogues for inhibition of inositol phosphate accumulation was L-PIA greater than adenosine greater than NECA, a finding indicating that A1-class adenosine receptors are involved in the inhibition. The reduction in inositol phosphate accumulation by L-PIA was blocked by an adenosine receptor antagonist, 8-phenyltheophylline. Stimulation of A1-class adenosine receptors inhibited isoproterenol-stimulated cyclic AMP accumulation as well as histamine-induced inositol phosphate accumulation. Both inhibitory effects were blocked by pretreatment of the cells with pertussis toxin [islet-activating protein (IAP)]. L-PIA also inhibited guanosine 5'-(gamma-thio)triphosphate (GTP gamma S)-stimulated accumulation of inositol phosphates in membrane preparations, and 8-phenyl-theophylline antagonized the inhibition. L-PIA could not inhibit GTP gamma S-induced accumulation of inositol phosphates in IAP-treated membranes. Gi/Go, purified from rabbit brain, inhibited GTP gamma S-stimulated accumulation of inositol phosphates in a concentration-dependent manner in membrane preparations. These results suggest that stimulation of A1-class adenosine receptors interacts with the IAP-sensitive G protein(s), resulting in the inhibitions of phospholipase C as well as adenylate cyclase in human astrocytoma cells.     

Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation.

We have previously shown that extracellular ATP acts as a mitogen via protein kinase C (PKC)-dependent and independent pathways (Wang, D., Huang, N., Gonzalez, F.A., and Heppel, L.A. Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells. I. Involvement of protein kinase C-dependent and independent pathways in the mitogenic response of mammalian cells to extracellular ATP. J. Cell. Physiol., 1991). The present aim was to determine if metabolism of arachidonic acid, resulting in prostaglandin E2 (PGE2) synthesis and elevation of cAMP levels, plays a role in mitogenesis mediated by extracellular ATP. Addition of ATP caused a marked enhancement of cyclic AMP accumulation in 3T3, 3T6, and A431 cells. Aminophylline, an antagonist of the adenosine A2 receptor, had no effect on the accumulation of cyclic AMP elicited by ATP, while it inhibited the action of adenosine. The accumulation of cyclic AMP was concentration dependent, which corresponds to the stimulation of DNA synthesis by ATP. The maximal accumulation was achieved after 45 min, with an initial delay period of about 15 min. That the activation of arachidonic acid metabolism contributed to cyclic AMP accumulation and mitogenesis stimulated by ATP in 3T3, 3T6, and A431 cells was supported by the following observations: (a) extracellular ATP stimulated the release of [3H]arachidonic acid and PGE2 into the medium; (b) inhibition of arachidonic acid release by inhibitors of phospholipase A2 blocked PGE2 production, cyclic AMP accumulation, and DNA synthesis activated by ATP, and this inhibition could be reversed by adding exogenous arachidonic acid; (c) cyclooxygenase inhibitors, such as indomethacin and aspirin, diminished the release of PGE2 and blocked cyclic AMP accumulation as well as [3H]thymidine incorporation in response to ATP; (d) PGE2 was able to restore [3H]thymidine incorporation when added together with ATP in the presence of cyclooxygenase inhibitors; (e) pertussis toxin inhibited ATP-stimulated DNA synthesis in a time- and dose-dependent fashion as well as arachidonic acid release and PGE2 formation. Other evidence for involvement of a pertussis toxin-sensitive G protein(s) in ATP-stimulated DNA synthesis as well as in arachidonic acid release is presented. In A431 cells, the enhancement of arachidonic acid and cyclic AMP accumulation by ATP was partially blocked by PKC down-regulation, implying that the activation of PKC may represent an additional pathway in ATP-stimulated metabolism of arachidonic acid. In all of these studies, ADP and AMP-PNP, but not adenosine, were as active as ATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The adenosine receptor mediated accumulation of cyclic AMP in Jurkat cells is enhanced by a lectin and by phorbol esters

The accumulation of cyclic AMP in Jurkat cells was stimulated by adenosine and adenosine analogues. The accumulation of cyclic AMP induced by these agents was competitively antagonized by the adenosine receptor antagonist 8-p-sulphophenyl-theophylline (KD appr 1.9 microM). The lectin PHA, the diacylglycerol OAG as well as tumor promoting phorbol esters enhanced the accumulation of cyclic AMP induced by the adenosine analogue NECA. The results suggest that activation of CD2/CD3 receptors by lectins could potentiate the endogenous cyclic AMP stimulator adenosine via activation of protein kinase C.     

Novel hydrolysis-resistant analogues of cyclic ADP-ribose: modification of the "northern" ribose and calcium release activity

Three novel analogues modified in the "northern" ribose (ribose linked to N1 of adenine) of the Ca(2+) mobilizing second messenger cyclic adenosine diphosphoribose, termed 2"-NH(2)-cyclic adenosine diphosphoribose, cyclic adenosine diphospho-carbocyclic-ribose, and 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, were synthesized (chemoenzymatically and by total synthesis) and spectroscopically characterized, and the pK(a) values for the 6-amino/imino transition were determined in two cases. The biological activity of these analogues was determined in permeabilized human Jurkat T-lymphocytes. 2"-NH(2)-cyclic adenosine diphosphoribose mediated Ca(2+) release was slightly more potent than that of the endogenous cyclic adenosine diphosphoribose in terms of the concentration-reponse relationship. Both compounds released Ca(2+) from the same intracellular Ca(2+) pool. In addition, the control compound 2"-NH(2)-adenosine diphosphoribose was almost without effect. In contrast, only at much higher concentrations (> or =50 microM) did the "northern" carbocyclic analogue, cyclic adenosine diphospho-carbocyclic-ribose, significantly release Ca(2+) from permeabilized T cells, whereas the previously reported "southern" carbocyclic analogue, cyclic aristeromycin diphosphoribose, was slightly more active than the endogenous cyclic adenosine diphosphoribose. Likewise, 8-NH(2)-cyclic adenosine diphospho-carbocyclic-ribose, expected to antagonize Ca(2+) release as demonstrated previously for 8-NH(2)-cyclic adenosine diphosphoribose, did not inhibit cyclic adenosine diphosphoribose mediated Ca(2+) release. This indicates that the 2"-NH(2)-group substitutes well for the 2"-OH-group it replaces; it may be oriented toward the outside of the putative cyclic adenosine diphosphoribose receptor binding domain and/or it can potentially also engage in H bonding interactions with residues of that domain. In sharp contrast to this, replacement of the endocyclic furanose oxygen atom by CH(2) in a carbocyclic system obviously interferes with a crucial element of interaction between cyclic adenosine diphosphoribose and its receptor in T-lymphocytes.     

Interleukin 2-dependent phosphorylation of interleukin 2 receptors and other T cell membrane proteins

The addition of IL 2 to Con A-activated splenic T cells induced the rapid and time-dependent phosphorylation of membrane proteins with m.w. of 115,000 to 105,000, 90,000, and 66,000, and to a lesser extent 55,000 to 58,000, 40,000, and 34,000. Immunoprecipitations conducted with an anti-IL 2 receptor antibody indicated that the murine IL 2 receptor (55,000 to 58,000) was included in the set of IL 2-dependent phosphoproteins. Phosphorylation of these same proteins was also seen after IL 2 treatment of PHA-activated T cells and of the IL 2-dependent line CTLL-2. Membrane phosphorylation was dependent on physiologically relevant IL 2 concentrations (0.2 to 1 ng/ml), and was detected as early as 1 min after IL 2 addition, with maximal levels of phosphorylation achieved by 15 min. In contrast to these observations, the pattern of cytoplasmic protein phosphorylation remained unchanged after IL 2 addition, although IL 2 did augment the level of preexisting cytoplasmic phosphorylation induced by lectin. The pattern of membrane protein phosphorylation induced by IL 2 also overlapped in part with that induced after stimulation of Con A-activated T cells with the phorbol ester PMA. IL 2-stimulated phosphorylation was inhibited by the addition of agents that both stimulate cyclic AMP-dependent protein kinases and block lymphocyte mitogenesis. No effect was seen upon addition of agents that enhance cyclic GMP-dependent protein kinases. These observations support a role for specific membrane as opposed to cytoplasmic protein phosphorylation in the regulation of lymphocyte growth by IL 2, and also suggest that protein kinase A, and perhaps protein kinase C, participate as regulators of the IL 2 signaling mechanism.     

Topography of protein kinases and phosphoproteins in the plasma membrane of 3T3 cells

The plasma membrane of 3T3 cells contains at least two different endogenous cyclic AMP-dependent protein kinase systems. One catalyzes the phosphorylation of endogenous protein substrates, i.e., PP24 and PP14, whereas the other catalyzes the phosphorylation of exogenous substrates. In this paper the topography of these cyclic AMP-dependent phosphorylation systems is described. The results show that the kinases which phosphorylate only exogenous substrates are primarily localized to the outer plasma membrane surface whereas the endogenous cyclic AMP-dependent protein kinase and its two endogenous substrates are localized to the cytoplasmic plasma membrane surface. The data also establish that neither the cytoplasmically orientated kinase nor its substrates has a transmembrane orientation even though factors acting on the outer plasma membrane can affect these proteins. This suggests that functional modulation of the cytoplasmically localized cyclic AMP-dependent phosphorylation system can be mediated by a transmembrane regulatory mechanism. The importance of determining the topography of such plasma membrane phosphorylation systems is emphasized by recent studies which show that neoplastic transformation can be mediated at least in part by protein kinases and/or phosphoproteins which are localized on the cytoplasmic surface of the plasma membrane.     

New locus (ttr) in Escherichia coli K-12 affecting sensitivity to bacteriophage T2 and growth on oleate as the sole carbon source.

The nature of resistance to phage T2 in Escherichia coli K-12 was investigated by analyzing a known phage T2-resistant mutant and by isolating new T2-resistant mutants. It was found that mutational alterations at two loci, ompF (encoding the outer membrane protein OmpF) and ttr (T-two resistance), are needed to give full resistance to phage T2. A ttr::Tn10 mutation was isolated and was mapped between aroC and dsdA, where the fadL gene (required for long-chain fatty acid transport) is located. The receptor affected by ttr was the major receptor used by phage T2 and was located in the outer membrane. Phage T2 was thus able to use two outer membrane proteins as receptors. All strains having a ttr::Tn10 allele and most of the independently isolated phage T2-resistant mutants were unable to grow on oleate as the sole carbon and energy source, i.e., they had the phenotype of fadL mutants. The gene fadL is known to encode an inner membrane protein. The most likely explanation is that fadL and ttr are in an operon and that ttr encodes an outer membrane protein which functions in translocating long-chain fatty acids across the outer membrane and also as a receptor for phage T2.     

Roles of lipopolysaccharide and outer membrane protein OmpC of Escherichia coli K-12 in the receptor function for bacteriophage T4

The roles of lipopolysaccharide and OmpC, a major outer membrane protein, in the receptor function for bacteriophage T4 were studied by using Escherichia coli K-12 strains having mutations in the ompC gene or in genes controlling different stages of lipopolysaccharide synthesis. The receptor activity for T4 was monitored by (i) T4 sensitivity of intact cells, (ii) phage inactivation activity of cell envelopes, and (iii) phage inactivation activity of specimens reconstituted from purified OmpC and lipopolysaccharide. It was found that (i) in the presence of the OmpC protein, the essential region of the lipopolysaccharide for the receptor activity was the core-lipid A region that includes the heptose region, whereas the glucose region was not necessarily required for the receptor function; (ii) the OmpC protein was not required at all when the distal end of the lipopolysaccharide was removed to expose a glucose residue at the distal end; and (iii) when cells lacked both the OmpC protein and the glucose region, they became extremely resistant to T4. Based on these findings, the roles of the OmpC protein and lipopolysaccharide in T4 infection are discussed.     

Adenosine potentiates lutropin-stimulated cyclic AMP production and inhibits lutropin-induced desensitization of adenylate cyclase in rat Leydig tumour cells.

The action of adenosine on lutropin (LH)-stimulated cyclic AMP production and LH-induced desensitization of adenylate cyclase in rat Leydig tumour cells was investigated. Adenosine and N6-(phenylisopropyl)adenosine caused a dose-dependent potentiation of LH-stimulated cyclic AMP production at concentrations (0.01-10 microM) which alone did not produce an increase in cyclic AMP production. However, 2-deoxyadenosine had no effect either alone or in combination with LH on cyclic AMP production. The potentiation produced by adenosine was unaffected by concentrations of the specific nucleoside-transport inhibitor dipyridamole, which inhibited [3H]adenosine uptake by up to 90%. The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine, but not RO-10-1724, inhibited the adenosine-induced potentiation. In the presence of adenosine, the kinetics of LH-stimulated cyclic AMP production were linear with time up to 2h, compared with those with LH alone, which showed a characteristic decrease in rate of cyclic AMP production after the first 15-20 min. Consistent with the altered kinetics, adenosine also inhibited the LH-induced desensitization of adenylate cyclase. These results suggest that adenosine has effects on rat tumour Leydig cells through receptors on the external surface of the plasma membrane. This receptor has characteristics similar to those of the R-type receptors, which have been shown either to stimulate or to inhibit adenylate cyclase. However, the effects of adenosine in the present studies does not involve a direct inhibition or activation of adenylate cyclase, but may involve an as yet undefined receptor-mediated modulation of adenylate cyclase.     

Functional Interaction of the tonA/tonB Receptor System in Escherichia coli

Host range mutants of phage T1 (T1h), which productively infected tonB mutants of Escherichia coli, were isolated. The phage mutants were inactivated by isolated outer membranes of E. coli in contrast to the wild-type phage, which only adsorbed reversibly. For the infection process, the tonB function is apparently only required for the irreversible adsorption of the phage T1, but not for the transfer of the phage DNA through the outer membrane and the cytoplasmic membrane of the cell. Mutants of the tonA gene expressing normal amounts of outer membrane receptor proteins were isolated and found to be partially sensitive to phage T5 and resistant to the phages T1 and T1h, colicin M, and albomycin and unable to take up iron as a ferrichrome complex. One tonA mutant remained partially sensitive to T5, colicin M, and albomycin and supported growth of T1h (not of T1) with the same plating efficiency as the parent strain. Only a small region of the tonA receptor protein seems to function for all the very different substrates. A newly isolated host range mutant of T5 (T5h) adsorbed faster to tonA(+) cells than did wild-type T5 and infected tonA missense mutants resistant to wild-type T5. The interplay of the tonA with the tonB function was observed with phage T5 infection, although T5 required only the tonA receptor. Ferrichrome inhibited plaque formation of T5 only when plated on tonB mutants. Adsorption of T5 to cells in liquid medium was influenced by ferrichrome as follows: complete inhibition by 0.1 muM ferrichrome with tonB mutants, not more than 35% inhibition by 1 to 100 muM ferrichrome with the tonB(+) parent strain in the presence of glucose as energy source, and 90% inhibition by 1 muM ferrichrome with partially starved parent cells. We conclude that there exist different functional states of the receptor protein that depend on the energy state of the cell and the tonB function. The latter seems to be required only for translocation processes with outer membrane proteins involved.     

Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.     

Aluminum fluoride induces phosphatidylinositol turnover, elevation of cytoplasmic free calcium, and phosphorylation of the T cell antigen receptor in murine T cells

Antigen activation of murine T lymphocytes leads to phosphorylation of three subunits of the murine T cell antigen receptor (L.E. Samelson, M.D. Patel, A.M. Weissman, J.B. Harford, and R.D. Klausner. 1986. Cell 46:1083). Two kinases are activated in this process: protein kinase C which leads to phosphorylation of the gamma and, to a lesser extent, the epsilon subunits on serine residues and a tyrosine kinase which phosphorylates the p21 subunit (M.D. Patel, L.E. Samelson, and R.D. Klausner. 1987. J. Biol Chem. 262:5831). We sought to determine whether treatment of these cells with NaF could simulate any of these antigen-induced events. Indeed NaF treatment resulted in breakdown of polyphosphoinositides and production of phosphoinositols. This treatment also resulted in a rise in cytosolic free Ca2+. EGTA failed to block this rise suggesting that NaF liberated intracellular stores of Ca2+. Finally NaF treatment resulted in phosphorylation of the gamma and epsilon chains of the T cell receptor indistinguishable from the effects of phorbol esters. The NaF effect was potentiated by addition of A1Cl3 consistent with the view that the active moiety is A1F4-. The A1F4--induced phosphorylations were abolished in cells in which protein kinase C was depleted by prior treatment with phorbol myristate acetate. All of these observations are compatible with the interpretation that the A1F4- phosphorylation is mediated by protein kinase C. Antigen and anti-receptor antibody-induced receptor serine phosphorylation and phophatidylinositol turnover are blocked by raising intracellular levels of cyclic adenosine monophosphate. In contrast, A1F4--induced effects were insensitive to cyclic adenosine monophosphate.     

Thyrotropin effects on thyroid cells in culture. Effects of trypsin on the thyrotropin receptor and on thyrotropin-mediated cyclic 3':5'-AMP changes.

Dog, human, and bovine thyroid cells in culture have been shown to develop follicle-like structures when cells are cultured in conditions of confluency and when cells are incubated in the presence of bovine thyrotropin or N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate during the first 24 to 48 hours after trypsinization. If thyrotropin is added 48 hours after trypsinization, these cells do not form follicle-like structures but remain as a monolayer culture. Although thyroid cells which grow as a monolayer have a thyrotropin receptor on their plasma membranes with the same in vitro binding properties as the thyrotropin receptor on the plasma membranes of the follicle-forming thyroid cells, there is a 1- to 2-fold greater number of receptors per mg of membrane protein when follicle-forming and monolayer cultures are compared...