pBFK, a new thermosensitive shuttle vector for Streptococcus pyogenes gene deletion by homologous recombination.

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a major human pathogen for which genetic manipulation remains an ongoing challenge. We created a new temperature-sensitive plasmid pBFK expressing spectinomycin resistance adapted to homologous recombination procedure to perform a complete gene deletion in GAS. Herein the mutagenesis strategy with pBFK was performed in a highly virulent GAS emm3 genotype.

Transcription, beta-like DNA polymerases and hypermutation.

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.

CD40-CD40L independent Ig gene hypermutation suggests a second B cell diversification pathway in humans.

Somatically mutated IgM(+)-only and IgM(+)IgD(+)CD27(+) B lymphocytes comprise approximately 25% of the human peripheral B cell pool. These cells phenotypically resemble class-switched B cells and have therefore been classified as postgerminal center memory B cells. X-linked hyper IgM patients have a genetic defect characterized by a mutation of the CD40L gene. These patients, who do not express a functional CD40 ligand, cannot switch Ig isotypes and do not form germinal centers and memory B cells. We report here that an IgM(+)IgD(+)CD27(+) B cell subset with somatically mutated Ig receptors is generated in these patients, implying that these cells expand and diversify their Ig receptors in the absence of classical cognate T-B collaboration. The presence of this sole subset in the absence of IgM(+)-only and switched CD27(+) memory B cells suggests that it belongs to a separate diversification pathway.

Two novel human and mouse DNA polymerases of the polX family.

We describe here two novel mouse and human DNA polymerases: one (pol lambda) has homology with DNA polymerase beta while the other one (pol mu) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix-loop-helix DNA-binding motifs and polymerase X domain. mRNA expression of pol lambda is highest in testis and fetal liver, while expression of pol mu is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol mu gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, gamma-rays or H(2)O(2)). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.

Early onset of immunoglobulin heavy chain gene rearrangements in normal human bone marrow CD34+ cells.

To characterize early B-cell precursors in humans, we correlated immunoglobulin heavy chain (IgH) gene rearrangement status with the CD34, CD19, and CD10 cell surface markers. Highly purified adult bone marrow (BM) cell fractions were obtained by two successive rounds of flow cytometric cell sorting, and IgH rearrangements were analyzed by polymerase chain reaction (PCR) amplification. Complete VDJH rearrangements were observed in the CD34+ CD19+ fraction, but not in the more immature CD34+ CD19- fraction. About one quarter of these rearrangements had an open reading frame, thus potentially permitting the synthesis of a mu chain. Partial DJH rearrangements were detected in both CD34+ CD19+ and CD34+ CD19- subsets, although they were less abundant in the latter. When triple labeling was used to better characterize the CD34+ CD19- population, DJH rearrangements were found to be present in the CD34(+) CD10+ CD19- fraction, but not in the more primitive CD34+ CD10- CD19-. These results indicate that IgH gene rearrangements occur in CD34+ BM cells and that they initiate in immature progenitors expressing the CD10, but not yet the CD19 surface antigen. Finally, the presence of IgH gene rearrangements in CD34+ BM cells provides a useful marker of clonality to evaluate the possible involvement of these cells in various B-cell lymphoid malignancies.

Convulxin, a potent platelet-aggregating protein from Crotalus durissus terrificus venom, specifically binds to platelets.

Convulxin, a very potent aggregating protein from rattlesnake venom, was purified by a new procedure and its heterodimeric structure alpha 3 beta 3 was confirmed. The polypeptide N-terminal sequences of convulxin subunits were determined by Edman degradation. They are very similar and appear homologous to botrocetin from Bothrops jararaca venom and to rattlesnake lectin from Crotalus atrox venom, both being classified among the C-type lectin family. The binding of 125I-labelled convulxin to blood platelets has also been analysed under equilibrium conditions. These studies indicated that convulxin binds to platelets with a high affinity (Kd = 30 pM) on a small number of binding sites (1000 binding sites per cell). The high-affinity binding of convulxin appears specific to platelets, since it is not observed on other cell types such as neutrophils and erythrocytes. Also, the high-affinity binding of convulxin to membranes platelet is not inhibited by alpha-thrombin, fibrinogen, collagen, laminin binding inhibitor, RGDS peptide, adenosine diphosphate, platelet-activating factor-acether, serotonin or epinephrine. This, together with the recent observation that platelet activation by convulxin is partially mediated by phospholipase C and involves other mechanisms as well, indicates that convulxin may interact with a specific platelet acceptor (receptor) protein which has yet to be characterized.

Dissociation between the phospholipases C and A2 activities in stimulated platelets and their involvement in the arachidonic acid liberation.

In previous work we have demonstrated that platelets depleted from secretory phospholipase A2 (sPLA2) produced similar amounts of thromboxane (Tx)B2 as control platelets upon stimulation by thrombin. However, since depletion of sPLA2 was not total, this sole finding only suggested the non-involvement of sPLA2 in arachidonic acid release. In the present study we provide further evidence for the non-involvement of sPLA2 in arachidonic acid liberation during platelet activation. Thus, rabbit platelets exposed to thrombin secreted sPLA2, released free arachidonic acid and formed TxB2 and inositol phosphates. In contrast, U46619, a stable prostaglandin (PG)H2 analogue, activates phospholipase C (PLC) and induces release of sPLA2 without TXB2 generation nor arachidonic acid liberation. At each concentration tested of both agonists, stimulation of sPLA2 activity paralleled the production of inositol phosphates. These data suggest that sPLA2 is dependent on phosphoinositide hydrolysis and on the release reaction and that it is not involved in the liberation of arachidonic acid from stimulated platelets. In addition, a dissociation was observed between sPLA2 and the enzyme involved in the arachidonic acid mobilization, suggesting that the liberation of this fatty acid from membrane phospholipids was mediated by cytosolic phospholipase A2 (cPLA2). Finally, PLC does not play a major role in arachidonic acid liberation, since U46619, which induced the breakdown of inositol phospholipids, failed to release arachidonic acid. In confirmation, neomycin, which inhibits PLC activity, failed to inhibit ATP, sPLA2 and arachidonic acid release upon stimulation of platelets by fluoroaluminate. These data demonstrate that sPLA2 is not involved in the arachidonic acid release by stimulated platelets and indicate that the activations of PLC, sPLA2 and cPLA2 are independent events.

Convulxin-induced platelet aggregation is accompanied by a powerful activation of the phospholipase C pathway.

Platelet aggregation and stimulation of phosphoinositide-specific phospholipase C (PLC) by thrombin and by convulxin (Cvx), a non-enzymic snake venom glycoprotein, were compared. Cvx-stimulated production of inositol phosphates by washed platelets was independent of the cyclo-oxygenase pathway, formation of platelet-activating factor and ADP release, but prostacyclin (prostaglandin I2), a stimulator of cyclic AMP formation, suppressed its effects on platelet and PLC activation. Kinetic analysis showed that inositol 1,4,5-trisphosphate formation reached its maximal value 15 s after platelet stimulation with Cvx and persisted for at least 5 min. Neomycin sulphate (10 mM), which complexes phosphatidylinositol 4-phosphate and phosphatidyl-inositol 4,5-bisphosphate, decreased the production of inositol phosphates, partially prevented platelet aggregation induced by a high concentration of Cvx (10 nM) and abolished both platelet aggregation and inositol phosphate formation induced by thrombin (2 units/ml) and by a stable prostaglandin H2 analogue, U46619 (1 microM). In contrast with neomycin sulphate, Na2SO4 had no significant effect against all agonists tested. It is concluded that platelet activation by Cvx is partially mediated by PLC and involves other mechanisms as well.

Secretory phospholipase A2 is not required for arachidonic acid liberation during platelet activation.

The subcellular localization of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in resting and activated platelets, and their involvement in arachidonic acid liberation during platelet activation, were studied. The amounts of sPLA2 and cPLA2 recovered were not modified during platelet activation. sPLA2 was mainly associated with the organelles of resting platelets (71% of total activity) and was released into the extracellular medium during cell activation (60% of total activity), whereas the majority of cPLA2 was localized in the cytosol of resting and activated platelets. The secretion of sPLA2 correlated with the release of ATP. sPLA2-depleted platelets aggregated as much as control platelets and produced similar amounts of thromboxane B2 upon thrombin activation. These results indicate that sPLA2 is not involved in the liberation of arachidonic acid during platelet activation.

Reduction by arachidonic acid of prostaglandin I2-induced cyclic AMP formation. Involvement of prostaglandins E2 and F2 alpha.

Arachidonic acid reverses the increase in cyclic AMP levels of washed human platelets exposed to prostaglandin (PG)I2, under conditions where the PGH2 analogue U46619 is ineffective. This effect of arachidonic acid was inhibited by aspirin, a cyclooxygenase inhibitor, but not by the thromboxane (Tx) synthase inhibitor Ridogrel, which induces, by inhibiting the conversion of PGH2 into TxA2, an overproduction of PGE2, PGD2 and PGF2 alpha. Addition of PGE2 or PGF2 alpha, which share a receptor with PGI2, to washed human platelets also induced a decrease in cyclic AMP levels, but PGD2, which interacts with a different receptor, had no effect. Thus neither PGD2, PGG2, PGH2, TxA2 nor TxB2 formed from arachidonic acid via the cyclooxygenase pathway is involved in the decrease in cyclic AMP levels. These findings were confirmed using forskolin, a diterpene from the labdane family, which enhanced the formation of cyclic AMP synergistically with the PGs. Also, arachidonic acid, unlike U46619, is able to reverse the inhibition of platelet aggregation by PGI2 after a lag phase of about 4 min. Our data indicate that arachidonic acid decreased cyclic AMP levels through its cyclooxygenase metabolites PGE2 and PGF2 alpha probably interacting competitively with the receptor of PGI2. In addition, intracellular cyclic AMP levels and the degree of aggregation of platelets by arachidonic acid seem to be inversely correlated.

Competitive inhibition of phospholipase A2 activity by the platelet-activating factor antagonist Ro 19-3704 and evidence for a novel suppressive effect on platelet activation.

The compound Ro 19-3704 [3-4(R)-2-(methoxycarbonyl) oxy-3-(octadecylcarbamoyl)oxy-propoxy butylthiazolium iodide], initially described as an antagonist of platelet-activating factor, is reported here to directly inhibit rabbit platelet phospholipase (PL) A2 activity, with an IC50 value of 4 to 7 microM. Classical Michaelis-Menten analysis showed that inhibition was reversible and competitive, inasmuch as apparent Km values increased in the presence of Ro 19-3704 (from 0.2-0.4 to 2 microM), whereas Vmax values remained constant (200 +/- 20 nmol/min/10(9) cells). Ro 19-3704 inhibited platelet aggregation, PLA2 release and thromboxane B2 formation induced by thrombin (0.25 U/ml), with IC50 values of 8, 15 and below 5 microM, respectively. Aggregation and PLA2 release by arachidonic acid (100 microM) were also inhibited, but thromboxane B2 formation was unaffected, indicating that Ro 19-3704 does not inhibit cyclooxygenase. Platelet activation by collagen (5 micrograms/ml), the thromboxane mimetic U46619 ([15(S)-hydroxy-11,9(epoxymethano)-prosta-5Z,13E-dienoic acid] 1 microM) and low concentrations of thrombin (0.05-0.1 U/ml) was also inhibited by Ro 19-3704. Inhibition of platelet activation was reversible, suggesting that its suppressive effect was not due to cytotoxicity. Finally, Ro 19-3704 did not stimulate cyclic AMP formation or inhibit phosphodiesterase activity. Ro 19-3704 is a competitive inhibitor of PLA2 activity, and is also endowed with a potent suppressive effect on platelet activation induced by different agonists.

Carrageenan-induced activation of human platelets is dependent on the phospholipase C pathway.

Stimulation of washed human platelets by the pro-inflammatory polysaccharide carrageenan is accompanied by shape change, aggregation and release of granule contents and unaccompanied by thromboxane A2 synthesis. Carrageenan triggers platelet activation through a prostaglandin synthetase-independent mechanism. The phospholipase A2 (PLA2) inhibitor, p-bromophenacyl bromide suppresses platelet responses to carrageenan (Vargaftig et al, 1980) probably by mechanism(s) other than those which involve PLA2 activity. Exposure of platelets to carrageenan (2-25 micrograms/ml) induced inositol phosphate formation in a time- and concentration-dependent manner, the level of inositol phosphate formation correlating with the intensity of aggregation. Neomycin, an aminoglycoside antibiotic which inhibits the phospholipase C-mediated phosphatidylinositol 4,5-bisphosphate breakdown, suppressed both platelet activation and inositol phosphate formation. Inhibition was concentration-dependent with an IC50 value of about 180 microM. Platelet-activating factor (PAF) is not responsible for carrageenan-induced platelet activation and inositol phosphate formation, since exposure of platelets to carrageenan (25 micrograms/ml) in the presence of compound WEB 2086 (100 microM), a PAF antagonist, failed to inhibit carrageenan responses. However, compound Ro 19-3704, a structurally related antagonist of PAF reported to be also an inhibitor of phospholipases A2 and C, inhibited concentration-dependently (0.1-10 microM) aggregation and ATP release induced by carrageenan (25 micrograms/ml). These findings indicate that carrageenan activates human platelets through a phospholipase C-dependent mechanism and show that neomycin, at low concentrations, can be a selective inhibitor of phospholipase C-mediated PIP2-breakdown.