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Initiation of large igneous provinces (LIPs) and temporal and spatial variations in their chemical compositions provide unique opportunities to understand mantle dynamics. It has been recently revealed that water‐rich reservoirs in the mantle sources have played a significant role in the production of LIPs. However, the dominant causes for the chemical variations in primitive magmas of LIPs remain debated. In the Emeishan LIP (ELIP), there are several places where picrites accompany more abundant flood basalts. These picrites span a large range of Ti/Y, Sm/Yb, and TiO2/Al2O3. In this work, we have determined the water contents of picrites with Ti/Y > 500 from the ELIP, based on the water content of clinopyroxene phenocrysts and water partitioning coefficients. We have also calculated the water content of the mantle sources by applying batch and fractional partial melting models. We show that the chemical compositions of picrites in the ELIP closely correlate with the calculated water content in their mantle sources. Our model suggests that the variation in the chemical composition of the primary magmas of the ELIP both on a local and regional scale can be mainly explained by the complementary roles of relatively dry recycled pyroxenite and hydrated components in the mantle plume, without requiring catastrophic thinning of the lithospheric mantle. Our model thus implies that both the initiation and chemical variation of the ELIP are affected by water from the deep mantle. Large igneous provinces (LIPs) represent a manifestation of the activity of a mantle plume. In most LIPs, the chemical compositions of basalts are heterogeneous and can usually be divided into high‐Ti and low‐Ti groups. This division has been used to infer differences in the mantle composition and/or partial melting conditions, which ultimately reflect the picture of the compositional and thermal structure of the deep mantle and their partial melting processes. In this study, we report that both the high‐Ti and low‐Ti type picrites of the Emeishan LIP were derived from hydrous mantle sources and such a compositional dichotomy is closely associated with the water content of the mantle sources. This case study provides a new perspective to connect the presence of volatiles in the deep mantle, the activity of deep‐seated mantle plumes, and widespread basaltic magmatism on the Earth's surface. Both high‐Ti and low‐Ti picrites in the Emeishan large igneous province are hydrousThe major and trace element compositions of picrites are related to the water content in their mantle sourcesCompositional variations in a large igneous provinces are affected by volatiles in mantle plumes Both high‐Ti and low‐Ti picrites in the Emeishan large igneous province are hydrous The major and trace element compositions of picrites are related to the water content in their mantle sources Compositional variations in a large igneous provinces are affected by volatiles in mantle plumes

Bordetella pertussis produces a calmodulin‐sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over‐produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti‐B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin‐dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post‐translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non‐pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi‐synthetic solid media. The frequency of these variants was between 10(‐6) and 10(‐7). About 250 variant strains were screened for the presence of virulence‐associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7‐11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5‐11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non‐random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.

The penetration of Bordetella pertussis adenylate cyclase into various mammalian cells exhibits similar kinetics; the accumulation of both intracellular cyclase activity and cyclic AMP is rapid, reaching constant levels after 15-60 min of incubation. The kinetics of enzyme penetration into turkey erythrocytes is different; cyclase activity and cyclic AMP accumulate linearly and do not reach constant levels even after 6 h of incubation. In the preceding paper [Friedman, Farfel & Hanski (1987) Biochem. J. 243, 145-151] we have suggested that the constant level of intracellular cyclase activity reflects a steady state formed by continuous penetration and intracellular inactivation of the enzyme. In contrast with other mammalian cells, no inactivation of cyclase is observed in turkey erythrocytes. These results further support the notion that there is continuous penetration and deactivation of the invasive enzyme in mammalian cells. A 5-6-fold increase in specific activity of the invasive cyclase is detected in a pellet fraction of human lymphocytes in which a similar increase in specific activity of the plasma-membrane marker 5′-nucleotidase is observed. A similar increase in the invasive-cyclase specific activity is detected in a membrane fraction of human erythrocytes. Cyclase activity in a membrane-enriched fraction of human lymphocytes reached a constant level after 20 min of cell exposure to the enzyme. Similar time courses were observed for accumulation of cyclase activity and cyclic AMP in whole lymphocytes [Friedman, Farfel & Hanski (1987) Biochem, J. 243, 145-151]. We suggest therefore that cyclic AMP generation by the invasive enzyme as well as the intracellular inactivation process occur while it is associated with a membrane fraction identical, or closely associated, with the plasma membrane.

Bordetella pertussis, the causative organism of whooping cough, produces a calmodulin-sensitive adenylate cyclase. Confer & Eaton [(1982) Science 217, 948-950] have shown that an extract from B. pertussis increases intracellular cyclic AMP levels in neutrophils and suggested that this increase is caused by the bacterial adenylate cyclase which penetrates these cells. We demonstrate in the present study that adenylate cyclase activity in lysates from lymphocytes exposed to a partially purified preparation of the bacterial enzyme has properties completely different from those of the intrinsic membrane-bound enzyme. Adenylate cyclase activity in lysates from lymphocytes exposed to the invasive enzyme is insensitive to N-ethylmaleimide, readily inactivated by acetic anhydride and relatively stable to SDS. Similar properties are exhibited by the bacterial enzyme itself. By contrast, the intrinsic membrane-bound enzyme activated by forskolin and guanosine 5′-gamma-thiotriphosphate is sensitive to N-ethylmaleimide and SDS and relatively stable to acetic anhydride. This strongly supports the notion that B. pertussis adenylate cyclase penetrates cells. Using the partially purified preparation of the invasive enzyme, we have studied the kinetics of its penetration. The intracellular catalytic activity reaches a steady state within 20 min, irrespective of enzyme or cell concentration. Steady-state levels are maintained for at least 2 h provided that the invasive enzyme is present in the incubation medium. Upon its removal, a rapid decrease (t1/2 approximately equal to 15 min) in the intracellular cyclase level is observed. This decrease reflects intracellular inactivation of the bacterial enzyme and is not caused by the release of the enzyme to the cell medium.

Pathogenic strains of Bordetella pertussis undergo spontaneous phase variation and become non‐pathogenic upon culturing in vitro. Spontaneous variants of the Tohama and #165 pathogenic strains of B. pertussis were selected by their ability to grow on synthetic and semi‐synthetic solid media. The frequency of these variants was between 10(‐6) and 10(‐7). About 250 variant strains were screened for the presence of virulence‐associated traits, such as production of hemolysin, pertussis toxin and filamentous hemagglutinin (FHA). Only four different combinations of the traits were found: 7‐11% of the variants displayed all traits, 17% of the variants carried the toxin and FHA, 5‐11% carried FHA only and 66% were devoid of all virulence traits. The strains which had at least one virulence trait also demonstrated some adenylate cyclase activity. The disappearance of hemolysin quantitatively affected the other traits. These results suggest that phase variation in B. pertussis is a non‐random process, involving multistep disappearance of virulence factors in the following order: hemolysin, pertussis toxin and FHA. In contrast, all 300 variants of strain #18323 of B. pertussis, which were able to grow on the selective solid media, carried all the virulence traits. This is in accordance with the strain's unique intracerebral growth capability.

Streptococcus pyogenes binds to the extracellular matrix (ECM) and a variety of host cells and tissues, causing diverse human diseases. Protein F, a S.pyogenes adhesin that binds fibronectin (Fn), contains two binding domains. A repeated domain (RD2) and an additional domain (UR), located immediately N‐terminal to RD2. Both domains are required for maximal Fn binding. In this study, we characterize RD2 and UR precisely and compare their functions and binding sites in Fn. The minimal functional unit of RD2 is of 44 amino acids, with contributions from two adjacent RD2 repeats flanked by a novel ‘MGGQSES’ motif. RD2 binds to the N‐terminal fibrin binding domain of Fn. UR contains 49 amino acids, of which six are from the first repeat of RD2. It binds to Fn with higher affinity than RD2, and recognizes a larger fragment that contains fibrin and collagen binding domains. Expression of UR and RD2 independently on the surface‐exposed region of unrelated streptococcal protein demonstrates that both mediate adherence of the bacteria to the ECM. We describe here a mechanism of adherence of a pathogen that involves two pairs of sites located on a single adhesin molecule and directed at the same host receptor.

The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A streptococcus. The hyaluronic acid capsule also appears to play an important role: studies of an acapsular mutant derived from the mucoid or highly encapsulated M protein type 18 group A streptococcal strain 282 indicated that loss of capsule expression was associated with decreased resistance to phagocytic killing and with reduced virulence in mice. To study directly the relative contributions to virulence of M protein and the hyaluronic acid capsule in strain 282, we inactivated the gene encoding the M protein (emm18) both in wild-type strain 282 and in its acapsular mutant, strain TX72. Inactivation of emm18 was accomplished by integrational plasmid mutagenesis, using the temperature-sensitive shuttle vector pJRS233 harboring a 5' DNA segment of emm18. As reported previously, wild-type strain 282 was resistant to phagocytic killing in vitro, both in whole human blood and in 10% serum. The capsule mutant TX72 was highly susceptible to phagocytic killing in 10% serum and moderately sensitive in whole blood. The M protein mutant 282KZ was highly susceptible to phagocytic killing in blood but only moderately sensitive in 10% serum. The double mutant TX74 was sensitive to killing in both conditions. In a mouse infection model, the 50% lethal dose was increased by 60- and 80-fold for the capsule and double mutants, respectively, compared with that of strain 282, but only by 6-fold for the M protein mutant. Integration of the strain 282 capsule genes into the chromosome of a nonmucoid M1 strain resulted in high-level capsule production and rendered the transformed strain resistant to phagocytic killing in 10% serum. These results provide further evidence that the hyaluronic acid capsule confers resistance to phagocytosis and enhances group A streptococcal virulence. The results suggest also that assessment of in vitro resistance to phagocytosis in 10% serum rather than in whole blood may be a more accurate reflection of virulence in vivo of group A streptococci.

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5′-[beta‐thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.

The pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) binds to fibronectin via protein F. In this study, we have investigated the binding properties of protein F to various multimeric tissue forms of fibronectin that appear on cell surfaces and in the extracellular matrix. We show that binding of S. pyogenes through protein F is more efficient to an in vitro-derived polymerized form of fibronectin (superfibronectin) than to soluble fibronectin immobilized in a solid phase. In addition, Chinese hamster ovary cells overexpressing the alpha5beta1 integrin produced an increased amount of a fibronectin matrix and consequently bound a higher number of S. pyogenes cells. Inhibition and direct binding assays using purified proteins demonstrated that binding to a fibronectin matrix involved both domains of protein F (UR and RD2) that have previously been implicated in interactions with fibronectin. Using intact S. pyogenes bacteria in which various domains of protein F were expressed as hybrids with the surface-exposed region of an unrelated protein, we revealed that, in contrast to the predominantly UR-mediated binding to soluble fibronectin, the maximal binding to the fibronectin matrix required RD2 in addition to UR. Since in some infections S. pyogenes may initially encounter a matrix form of fibronectin, these results suggest that UR and RD2 may be important for the initiation of streptococcal infectious processes.

The existence of an invasive adenylate cyclase in dialyzed urea extracts of the bacterium Bordetella pertussis has been suggested recently. Gel filtration of B. pertussis dialyzed urea extract shows that the invasive enzyme constitutes only a small portion of the total adenylate cyclase activity found in the extract. Its size is different than the size of the two peaks of adenylate cyclase activity identified in the extract. Ca2+ is absolutely required for the penetration of the invasive enzyme, it also controls the rate of intracellular cAMP accumulation in human lymphocytes exposed to dialyzed extract. These characteristics may be attributed to the increase in the size of the invasive enzyme as found by gel filtration chromatography of the extract in the absence of Ca2+. Removal of nonpenetrating adenylate cyclase that adheres to lymphocytes permits a direct assay of the intracellular enzyme. The time course of intracellular accumulation of adenylate cyclase activity is similar to the time course of intracellular accumulation of cAMP, suggesting that the invasive enzyme is rapidly deactivated, but not degraded, since it can be detected upon cell disruption. No appreciable amount of the enzyme is introduced when cells are incubated with extract at 4 degrees C for 120 min, then washed and incubated further at 37 degrees C. Concanavalin A inhibits cAMP accumulation irrespective of the time of its addition, and EGTA prevents penetration of the invasive enzyme even if added 20 min after addition of extract. These findings are different from those observed in other bacterial toxins thought to be internalized by receptor-mediated endocytosis. However, the cellular penetration of B. pertussis adenylate cyclase is cell-selective. It does not occur in human erythrocytes. In addition to human lymphocytes, S49 cyc- murine lymphoma, turkey erythrocytes, and rat oocytes accumulate cAMP in response to B. pertussis extract.

Bordetella pertussis produces extracytoplasmic adenylate cyclase toxin (AC toxin) which penetrates target cells and, upon activation by host calmodulin, generates high levels of intracellular cyclic AMP (cAMP). As a result, bactericidal functions of immune effector cells are impaired. Since a considerable amount of AC toxin is associated with the bacterium, it was proposed that the toxin may be delivered by direct interaction of the organism with the target cells (E. L. Hewlett, M. C. Gray, and R. D. Pearson, Clin. Res. 35:477A, 1987). Incubation of CHO cells with intact B. pertussis led to formation of intracellular cAMP at levels comparable to those produced in CHO cells by equivalent activities of isolated AC toxin. cAMP accumulation induced by the whole bacteria appeared after a lag of 40 to 60 min and reached high levels within 2 to 3 h, whereas adherence of the bacteria proceeded rapidly and reached a maximal level within 80 min. Sera of pertussis patients completely blocked cAMP accumulation induced by the whole bacteria without having a major effect on either bacterial adherence or cAMP production by the AC toxin. Cytochalasins B and D, inhibitors of bacterial invasion, abrogated the cAMP response to the whole bacteria but not the response to the AC toxin. These agents did not affect bacterial adherence. Transmission electron micrographs revealed that B. pertussis, within the time course of cAMP induction, invaded CHO cells. We suggest that cAMP induction by B. pertussis is caused by the entry of the whole bacteria into CHO cells rather than by delivery of AC toxin during bacterial adherence. This route of cell intoxication may be relevant to the pathogenesis of whooping cough.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the ‘dense-vesicle’ cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and ‘dense-vesicle’ cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.

In a previous study we reported the identification of protein F, a fibronectin-binding protein that was essential to the ability of Streptococcus pyogenes JRS4 to adhere to respiratory epithelial cells (E. Hanski and M. Caparon, Proc. Natl. Acad. Sci. USA, 89:6172-6176, 1992). To further evaluate the role of protein F in the adherence of the group A streptococci, we screened other group A streptococcal strains, including six recent clinical isolates, and one strain of Enterococcus faecalis for their capacity to bind fibronectin and for the presence of the gene encoding protein F (prtF). Seven of eight group A streptococcal strains analyzed, including all recent clinical isolates, both bound fibronectin at high affinity and contained DNA sequences that hybridized with a prtF-specific probe. One group A streptococcal isolate and the strain of E. faecalis examined neither contained a prtF-related gene nor bound fibronectin. These two strains also could not efficiently adhere to respiratory epithelial cells. However, upon the introduction of the cloned prtF gene, both of these strains gained the capacity to bind fibronectin and to adhere to respiratory epithelial cells. These results suggest that protein F is an important adhesin, which may have a general role in the virulence of the group A streptococci.

Two forms of Bordetella pertussis adenylate cyclase of 200 and 47 kDa have been purified from dialyzed urea extract of the bacteria to specific activities of 466 and 1685 mumol.min-1.mg-1, respectively. Both forms are activated 50-200-fold by calmodulin. The half-maximum concentration required for the activation of the 200-kDa catalyst is 5.4.10(-9) M, whereas the one required for activation of the 47-kDa catalyst is 1.8.10(-10) M. Polyclonal antibodies raised against the 47-kDa catalyst specifically recognize both forms of the enzyme in purified state as well as in bacterial extracts on immunoblots. The antibody inhibits at similar titer adenylate cyclase activity of the purified forms as well as the activity present in dialyzed urea extract of the bacteria. It also prevents the penetration of the invasive B. pertussis adenylate cyclase into human lymphocytes. The inhibition induced by the antisera is specific to B. pertussis enzyme, since both calmodulin-dependent brain and sperm adenylate cyclase are not affected by the antibody.

Bordetella pertussis produces a calmodulin‐sensitive adenylate cyclase (AC) which is an essential virulence factor in mammalian pertussis. Here we report the purification and characterization of the toxic form of the enzyme, which penetrates eukaryotic cells and generates high levels of intracellular cAMP. This form was purified from an extract of B.pertussis strain carrying a recombinant plasmid which over‐produced both enzymatic and toxic activities of the enzyme. Western blot analysis of the extract using anti‐B.pertussis AC antibodies detected only one protein of 200 kd. However, gel filtration of the extract resolved two peaks of enzymatic activity. The first peak of aggregated material contained greater than 70% of the total enzymatic activity, and the second peak contained the majority of the toxic activity. Purification of the enzyme from both peaks yielded proteins of 200 kd, with similar biochemical and immunological properties. Yet only the enzyme purified from the second peak could penetrate human lymphocyte and catalyse the formation of intracellular cAMP. B.pertussis AC gene expressed in Escherichia coli produced a calmodulin‐dependent enzyme of 200 kd, which lacked lymphocyte penetration capacity. It is proposed that a post‐translational modification that occurs in B.pertussis but not in E.coli confers upon the 200 kd protein of B.pertussis AC the toxic properties.

Binding to fibronectin has been suggested to play an important role in adherence of the group A streptococcus Streptococcus pyrogenes to host epithelial cells; however, the identity of the streptococcal fibronectin receptor has been elusive. Here we demonstrate that the fibronectin-binding property of S. pyogenes is mediated by protein F, a bacterial surface protein that binds fibronectin at high affinity. The gene encoding protein F (prtF) produced a functional fibronectin-binding protein in Escherichia coli. Insertional mutagenesis of the cloned gene generated a mutation that resulted in the loss of fibronectin-binding activity. When this mutation was introduced into the S. pyrogenes chromosome by homologous recombination with the wild-type allele, the resulting strains no longer produced protein F and lost their ability to bind fibronectin. The mutation could be complemented by prtF introduced on a plasmid. Mutants lacking protein F had a much lower capacity to adhere to respiratory epithelial cells. These results demonstrate that protein F is an important adhesin of S. pyogenes.

The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A streptococcus. The hyaluronic acid capsule also appears to play an important role: studies of an acapsular mutant derived from the mucoid or highly encapsulated M protein type 18 group A streptococcal strain 282 indicated that loss of capsule expression was associated with decreased resistance to phagocytic killing and with reduced virulence in mice. To study directly the relative contributions to virulence of M protein and the hyaluronic acid capsule in strain 282, we inactivated the gene encoding the M protein (emm18) both in wild-type strain 282 and in its acapsular mutant, strain TX72. Inactivation of emm18 was accomplished by integrational plasmid mutagenesis, using the temperature-sensitive shuttle vector pJRS233 harboring a 5' DNA segment of emm18. As reported previously, wild-type strain 282 was resistant to phagocytic killing in vitro, both in whole human blood and in 10% serum. The capsule mutant TX72 was highly susceptible to phagocytic killing in 10% serum and moderately sensitive in whole blood. The M protein mutant 282KZ was highly susceptible to phagocytic killing in blood but only moderately sensitive in 10% serum. The double mutant TX74 was sensitive to killing in both conditions. In a mouse infection model, the 50% lethal dose was increased by 60- and 80-fold for the capsule and double mutants, respectively, compared with that of strain 282, but only by 6-fold for the M protein mutant. Integration of the strain 282 capsule genes into the chromosome of a nonmucoid M1 strain resulted in high-level capsule production and rendered the transformed strain resistant to phagocytic killing in 10% serum. These results provide further evidence that the hyaluronic acid capsule confers resistance to phagocytosis and enhances group A streptococcal virulence. The results suggest also that assessment of in vitro resistance to phagocytosis in 10% serum rather than in whole blood may be a more accurate reflection of virulence in vivo of group A streptococci.

Bordetella pertussis produces a calmodulin-dependent adenylate cyclase (AC) which acts as a toxin capable of penetrating eukaryotic cells and generating high levels of intracellular cyclic AMP. Transfer of target cells into B. pertussis AC-free medium leads to a rapid decay in the intracellular AC activity, implying that the invasive enzyme is unstable in the host cytoplasm. We report here that treatment of human lymphocytes with a glycolysis inhibitor and an uncoupler of oxidative phosphorylation completely blocked the intracellular inactivation of B. pertussis AC. Lymphocyte lysates inactivated all forms of B. pertussis AC in the presence of exogenous ATP. This inactivation was associated with degradation of an 125I-labelled 200 kDa form of B. pertussis AC. It appears that ATP is required for the proteolytic pathway, but not as an energy source, since non-hydrolysable ATP analogues supported inactivation and complete degradation of the enzyme. The possibility that binding of ATP to B. pertussis AC renders it susceptible to degradation by the host cell protease is discussed.

Rat oocytes resume meiosis spontaneously in vitro within 3 h after their isolation from the ovarian follicles. We report here that the spontaneous maturation of isolated rat oocytes is preceded by a drop in intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP). Further experiments were carried out to examine the possible correlation between the meiotic status and cAMP levels within the oocyte. To challenge rat cumulus-free oocytes to generate cAMP, bypassing their own adenylate cyclase, a preparation of an invasive adenylate cyclase from Bordetella pertussis was used. We found a dose-dependent elevation of cAMP levels within these oocytes that corresponded to inhibition of their spontaneous maturation. Persistent inhibition of meiosis was obtained with the continuous presence of the enzymatic preparation, whereas its removal resulted in a transient inhibition associated with a drop in cAMP. We suggest that the presence of elevated cAMP levels in the oocyte is directly responsible for the maintenance of meiotic arrest.

ATLAS, HANSKI AND LEVITZKI REPLY—Our reply will be divided into two parts: first, the validation of our results1, and second, the demonstration that the methodology used by Pochet and Schmitt is inadequate.