Coiled-coil domains in proteins secreted by type III secretion systems

STUDY OBJECTIVE: To evaluate the immediate cytologic assessment during CT-guided fine-needle aspiration cytology (FNAC) in the diagnosis of operable indeterminate solitary pulmonary nodules (SPNs). DESIGN: Prospective randomized study. PATIENTS AND METHODS: Two hundred twenty patients with SPN undergoing CT-guided FNAC were divided into two groups. In the first one (group A, 110 patients), a cytologist assessed the adequacy of the sample obtained immediately, and when the sample was considered inadequate, fine-needle aspiration (FNA) was repeated. In the second group (B, 110 patients), an immediate cytologic examination was not performed, but only a gross assessment by the surgeon. Histologic study of the SPN was possible in 217 cases, whereas three patients were followed up radiologically. RESULTS: Adequate samples were obtained in 100% of group A and 88% of group B (p<0.001). The diagnostic accuracy was 99% in group A and 81% in group B (p<0.001). Group A required a mean of 1.22 FNAs compared with 1.10 in group B (p=0.015). The rate of pneumothorax in the whole series was 24%, and statistically significant differences between the two groups were not detected. CONCLUSIONS: Immediate cytologic study significantly increased the adequacy and diagnostic accuracy of CT-guided FNAC of indeterminate SPNs without causing a significant increase of complications.     

Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides

Pseudomonas aeruginosa elastase and the LasA protease are synthesized as preproenzymes with long amino-terminal propeptides. The elastase propeptide is cleaved autocatalytically in the periplasm to form a transient, inactive elastase-propeptide complex. In contrast, the processing of proLasA does not involve autoproteolysis. In this study, we analyzed short-term P. aeruginosa cultures under conditions that minimize proteolysis and found that an elastase-propeptide complex is secreted, and then the propeptide is degraded extracellularly, apparently by elastase itself. LasA protease, on the other hand, was found to be secreted in its unprocessed 42-kDa proenzyme form. The processing of proLasA occurred extracellularly, and it involved the transient appearance of a 28-kDa intermediate and the respective 14-kDa LasA propeptide fragment. The processing of proLasA in P. aeruginosa strain FRD740, which does not express elastase, also proceeded via the 28-kDa intermediate, but the rate of processing was greatly reduced. This low rate of proLasA processing was further reduced when the activity of a secreted lysine-specific protease was blocked. Purified secreted proteases of P. aeruginosa (i.e. elastase, the lysine-specific protease, and alkaline proteinase) converted proLasA to the active enzyme. Processing by elastase and the lysine-specific enzyme, but not by alkaline proteinase, proceeded via the 28-kDa intermediate, and both were far more effective than alkaline proteinase in converting proLasA to the mature enzyme. We conclude that LasA protease and elastase are secreted with their propeptides, which are then degraded by secreted proteases of P. aeruginosa. In addition to their other functions, the propeptides may play a role in targeting their respective enzymes across the outer membrane.     

Metabolites from an Antarctic sponge-associated bacterium, Pseudomonas aeruginosa

In an ongoing survey of the bioactive potential of microorganisms associated with marine invertebrates, the culture media of a sponge-associated bacterial strain of Pseudomonas aeruginosa was found to contain metabolites which inhibit the growth of several Gram-positive microorganisms. A series of diketopiperazines (1-6) including a new natural product (6) and two known phenazine alkaloid antibiotics (7 and 8) were isolated from the culture broth of this bacterium.     

Structure of adenylate cyclase and the coupling with the receptor-G protein system

Adenylate cyclase is a key enzyme that couples with both the stimulatory and inhibitory G proteins (Gs and Gi). The cyclase has been purified and shown to be a glycoprotein of molecular weight 115,000-180,000. Cloning of cDNAs for adenylate cyclase showed that the cyclase is a member of a large family consisting of a variety of subtypes of the enzyme. These subtypes show different responses to calmodulin and G protein beta gamma subunits, and their distributions in tissues and organs are also different. This suggests that each subtype is involved in a particular physiological function. The general structure of adenylate cyclase is composed of two cytoplasmic domains and two membrane-spanning domains, each of which contains 6 transmembrane spans (12 spans in a molecule). The amino acid sequence of each cytoplasmic domain, which is thought to contain a nucleotide (ATP) binding site, is well-conserved among the various subtypes. This review also focuses on the regulation of adenylate cyclase activity by G protein subunits, particularly on several models for adenylate cyclase inhibition by Gi. As one of these mechanisms, direct inhibition of adenylate cyclase by the beta gamma subunits recently demonstrated by us will be discussed.     

Arthritis in burned persons, caused by Pseudomonas aeruginosa

The clinical course and morphological changes in 33 limb joints in 20 burnt patients with pyocyanic arthritis have been studied. The latter is characterized by a grave clinical course and not infrequently complicated with sepsis which becomes especially hazardous after destruction of articular cartilages. Destructive changes in the joints would develop in different terms: following 25-45 days in large joints, in smaller one 10-15 days after the appearance of the clinical picture of this complication. Surgical therapy (arthrotomy, amputation of the extremity) associated with conservative measures (antibacterial therapy, blood transfusion, etc) should be considered to be the mostly effective treatment for destructive forms of arthritis.     

Acetylation of amikacin, a new semisynthetic antibiotic, by Pseudomonas aeruginosa carrying an R factor

A clinical isolate Pseudomonas aeruginosa GN315 resistant to amikacin (AK), a new semisynthetic antibiotic, inactivated AK by acetylation. The acetylating enzyme was purified approximately 146-fold from a crude extract of GN315 by affinity chromatography. Fractionated samples obtained by affinity chromatography showed almost the same inactivation curves toward 3',4'-dideoxykanamycin B (DKB) and AK. Partially purified AK-acetylating enzyme inactivated DKB and kanamycin A but could not inactivate gentamicin C(1). The optimal pH for their inactivation was 6.0 to 7.0, and the pH curves for the inactivation of both drugs were almost the same. These facts indicate that AK and DKB are inactivated by the same aminoglycoside-acetylating enzyme. Through elemental analysis, the inactivated AK was found to be a monoacetylated product of AK. A sample of inactivated AK was purified and compared with a synthetic 6'-N-acetyl AK by thin-layer chromatography, and the results indicated that AK was inactivated by acetylation of the 6'-NH(2) group. The ultraviolet, infrared, and nuclear magnetic resonance spectra of the inactivated AK showed that AK was inactivated by the enzyme through acetylation of the amino group of 6'-amino-6'-deoxy-d-glucose moiety of AK. This enzyme, mediated by R factor, is capable of conferring resistance to AK, DKB, kanamycin, gentamicin, and sulfanilamide.     

Synergistic activation of adenylate cyclase by guanylyl imidophosphate and epinephrine

A kinetic analysis of the synergistic activation of turkey erythrocyte adenylate cyclase by 1-catecholamines and guanylyl imidodiphosphate (Gpp(NH)p) is described. We have found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide according to the following mechanism: R-E+G=R-EG R-EG+H=HR-EG leads to HR-E'G where R is the receptor, E the enzyme, G the guanyl nucleotide effector, and H the hormone. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active stable form (E') occurs with a rate constant of k=0.7 min-1. This step is essentially irreversible in the presence of high Gpp(NH)p concentrations. In the absence of beta-agonist (1-catecholamine) and at low free Mg2+ concentrations, the activation of the enzyme is insignificant. At high Mg2+ concentration the conversion of E to E' occurs slowly in the absence of hormone, probably by another pathway. Thus, the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. The process of enzyme activation requires both Gpp(NH)p and hormone and under these conditions is essentially irreversible. The permanently active enzyme is stable in the absence of hormone and Gpp(NH)p and its high catalytic activity is stable for many hours. However, hormone and ATP induce a conversion of the high activity to the low activity form. Thus, it seems that both the process of enzyme activation by Gpp(NH)p and its reversal are hormone dependent. Both processes are blocked by the beta-blocker propranolol.     

Regulation of adenylate cyclase coupled beta-adrenergic receptors by beta-adrenergic catecholamines

Injection of frogs with beta-adrenergic catecholamines produced a selective desensitization (loss of responsiveness) of the erythrocyte membrane adenylate cylase to subsequent stimulation in vitro by isoproterenol. Basal, prostaglandin E1- and fluoride-sensitive enzyme activities were unaffected. A 77% (p less than 0.001) decline in isoproterenol-responsive enzyme activity in the cells from the treated animals was observed with no change in the Km for isoproterenol stimulation of the enzyme (concentration causing 1/2 maximal enzyme activation). The decrease in catecholamine-sensitive adenylate cyclase was accompanied by a parallel 68% (p less than 0.001) fall in the apparent number of beta-adrenergic receptors in the erythrocyte membranes, assessed by (-) (3H)alprenolol binding studies. There was no change in the affinity of the receptor binding sites. The catecholamine-induced desensitization and fall in the beta-adrenergic receptor number were both concentration and time-dependent and displayed beta-adrenergic specificity. Isoproterenol was more potent in desensitizing cells and in lowering the receptor number than was norepinephrine. The beta-adrenergic antagonist propranolol, but not the alpha-adrenergic antagonist phentolamine, blocked the desensitizing effects of isoproterenol. Propranolol itself, however, did not cause desensitization. Cells became resensitized to the stimulatory effects of catecholamines, in association with a return in beta-receptor number, when propranolol was injected into previously desensitized animals. The changes in receptor number in membranes from desensitized and resensitized animals were also reflected in soluble receptor preparations. The protein synthesis inhibitor cycloheximide did not affect either desensitization, resensitization, or the changes in receptor number which accompanied the changes in adenylate cyclase sensitivity to catecholamines. These findings suggest that the chronic occupancy of beta-adrenergic receptors by beta-adrenergic agonists (but not antagonists) decreases the number of functional beta-adrenergic receptor binding sites and, hence, lowers the responsiveness of adenylate cylase to catecholamine stimulation. The lack of effort of cycloheximide on these regulatory effects suggests that "inactivation" and subsequent "reactivation" of the receptors, rather than changes in receptor turnover, are involved.     

Inhibition of adenylate cyclase of rat hepatic membranes by glycosaminoglycans

Glycosaminoglycans are long non-branched polysaccharides composed of repeating disaccharide units. In a previous in vitro study we have shown that such molecules are able to modulate substrate phosphorylation catalyzed by cAMP-dependent protein kinase. Here, we investigate the impact of glycosaminoglycans, such as heparan sulfate, dermatan sulfate, chondroitin 4- and 6-sulfate, keratan sulfate and hyaluronic acid upon adenylate cyclase, which directly regulates cAMP-dependent protein kinase activity via cAMP synthesis. In rat liver plasma membrane preparation we have determined forskolin- and guanosine-5'-beta, gamma-imidotriphosphate-induced cAMP formation catalyzed by adenylate cyclase in the presence of increasing concentrations of glycosaminoglycans. The results indicate that glycosaminoglycans strongly influence enzymic conversion of ATP into cAMP. The highest reduction of adenylate cyclase activity is observed in the presence of dermatan sulfate and heparan sulfate. Moreover, the inhibitory effect of these two glycosaminoglycans is higher when guanosine-5'-beta, gamma-imidotriphosphate, instead of forskolin, is used as stimulator of adenylate cyclase. Further characterization of enzyme inhibition mediated by dermatan sulfate shows that this molecule exerts an inhibitory effect of mixed type.     

Pneumonia caused by coliforms and Pseudomonas aeruginosa

The diagnosis and treatment of 20 hospital patients seen in the past year with proven pneumonia caused by coliforms and Pseudomonas aeruginosa are discussed. Predisposing factors and methods for improving laboratory and clinical diagnosis are analysed, the main problem being to discriminate between genuine pneumonia caused by these organisms and mere contamination of sputum samples resulting from colonization of the upper respiratory tract following broad-spectrum chemotherapy. Overall initial chemotherapy with gentamicin cured 75% (15 out of 20) of the patients in spite of unfavourable underlying pathology. Where gentamicin was given in adequate dosage, which in practice meant that dose which produced peak serum concentrations of 8 mug/ml or more, the cure rate was 91% (11 out of 12). In those patients achieving (measured) peak serum concentrations of less than 8 mug/ml the cure rate was only 33% (4 out of 12). These figures include four patients who failed to respond to doses of gentamicin producing peak concentrations of 5-0-6-0 mug/ml in each case. These patients responded promptly to higher doses (or accumulation), producing peak serum concentrations of 8 mug/ml or more and were then cured within three to five days. Toxicity from gentamicin was not observed in any patient. These results indicate that it is necessary to monitor gentamicin therapy by laboratory assay to ensure adequate dosage and that peak serum concentrations of 8 mug/ml or more are significantly correlated with successful treatment of pneumonia caused by coliforms and Ps. aeruginosa.     

Bradykinin generation triggered by Pseudomonas proteases facilitates invasion of the systemic circulation by Pseudomonas aeruginosa

To elucidate the mechanism of bacterial exoprotease in promotion of the intravascular dissemination of Pseudomonas aeruginosa, we examined the possible involvement of bradykinin (whose generation is induced by pseudomonal proteases in septic foci) in the invasion by bacteria, and in access of bacterial toxins to systemic blood circulation. P. aeruginosa 621 (PA 621), which produces very little protease, was injected intraperitoneally into mice together with pseudomonal exoproteases (elastase/alkaline protease). Dissemination of bacteria from the peritoneal septic foci to the blood was assessed by counting viable bacteria in the blood and spleen by use of the colony-forming assay. The results showed that pseudomonal proteases markedly enhanced (10- to 100-fold) intravascular dissemination of bacteria in mice. This enhancement was induced not only by pseudomonal proteases but also by bradykinin. More importantly, the increased spread of PA 621 induced by pseudomonal protease and bradykinin was significantly augmented by the addition of kininase inhibitors, indicating the direct involvement of bradykinin in bacterial dissemination. Similarly, bradykinin caused effective dissemination of pseudomonal toxins such as endotoxin (lipopolysaccharide) and exotoxin A when the toxins were injected into the peritoneal cavity with bradykinin. Furthermore, the lethality of the infection with PA 621 was strongly enhanced by pseudomonal proteases given i.p. simultaneously with PA 621. On the basis of these results, it is strongly suggested that pseudomonal proteases as well as bradykinin generated in infectious foci are involved in facilitation of bacterial dissemination in vivo.     

Antibiotic resistance in Pseudomonas aeruginosa: an Italian survey

In order to assess the current level of resistance to widely used antipseudomonal antibiotics in clinical isolates of Pseudomonas aeruginosa, a national survey was undertaken. Fifteen hospitals throughout Italy participated in the study. The University of Catania tested the antibiotic susceptibility of 1005 consecutive clinically significant P. aeruginosa collected from March to June 1995. Lack of susceptibility, according to NCCLS breakpoints, was at the following rates: meropenem, 9.1%; imipenem, 19.3%; ceftazidime, 13.4%; carbenicillin, 27.3%; piperacillin, 12%; ticarcillin/clavulanic acid, 22.8%; amikacin, 10.6%; and ciprofloxacin, 31.9%. About half of the isolates (44.4%) were not susceptible to at least one of the antibiotics tested.     

Activation by cholera toxin of adenylate cyclase solubilized from rat liver

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.     

Regulation of adenylate cyclase from cultured human cell lines by adenosine

Broken cell particulate preparations of adenylate cyclase isolated from the human glioma cell line 132-1N1 were stimulated 2-to 3-fold by 30 muM adenosine. This concentration of adenosine produced a maximal stimulation of the cyclase while 3 to 5 muM adenosine produced half-maximal stimulation. Theophylline, at 40 muM, inhibited the adenosine stimulation of the adenylate cyclase by about 40% while 200 muM produced near complete inhibition. The inhibition by theophylline could be overcome by increasing adenosine to a concentration 10-fold that of theophylline, implying that the inhibition was competitive. Basal activity was not inhibited by even 1.0 mM theophylline, nor was the epinephrine stimulated activity. In contrast, 1.0 muM propranolol essentially completely inhibited the 8-fold stimulation of 1.0 muM epinephrine but had no effect on either basal or adenosine-stimulated activity. Adenosine and 2-chloroadenosine were equipotent in stimulating adenylate cyclase from the 132-1N1 line, whereas neither adenine nor guanosine had any detectable effect. GTP, 10 muM, produced a small variable stimulation of the adenylate cyclase while the GTP analogue, 5'-guanylylimidodiphosphate (Gpp(NH)p), produced a marked stimulation fo the cyclase. Preincubation of the adenylate cyclase preparation with the analogue greatly increased its potency and maximal effect. In contrast, both basal and adenosine-stimulated activity decreased markedly with preincubation. The effects of adenosine or epinephrine in combination with Gpp(NH)p were at least additive and often synergistic in comparison to the effects of the compounds alone. The effects of adenosine on intact and broken cell preparations of the human fibroblast lines WI-38 and VA13-2RA were also examined. In the intact VA13-2RA, adenosine produced rapid and large increases in intracellular and extracellular cyclic adenosine 3':5'-monophosphate (cAMP). In the parental fibroblast line, the WI-38, adenosine slightly elevated basal levels of cAMP, but only produced marked elevations in the presence of non-methylxanthine phosphodiesterase inhibitors. The effect of adenosine on the broken cell particulate preparations of adenylate cyclase from the fibroblasts was similar to its action on the cyclase from the 132-1N1; 30 muM adenosine produced a maximal stimulation of the adenylate cyclase, and the stimulation was inhibited by theophylline.     

Cardiac beta-adrenoceptors, G-proteins and adenylate cyclase regulation during myocardial hypertrophy

Reactivity to Alcohol and Neutral Cues was compared in male, inpatient problem drinkers (N = 30). Subjects reported an overall desire not to drink alcohol which decreased in the presence of the Alcohol Cue. There were no cue-specific changes in heart rate, blood pressure and arousal level and a non-significant trend for increased stress in the Alcohol Cue condition. Subgroups of subjects reporting positive and negative desire for alcohol were identified. Subjects who reported negative desire for alcohol in the Alcohol Cue condition also reported greater self-efficacy to resist drinking in the future. These findings have implications for understanding the nature of self-reported desire for alcohol and its potential role in the treatment of alcohol dependence.     

An EGF receptor-mediated signal attenuates the inhibitory effect of LPA on an adenylate cyclase activity

A tyrosine kinase receptor-mediated and a heterotrimeric G protein-coupled receptor-mediated signals have been shown to evoke distinct intracellular signaling events. There has been increasing evidence that cross-talk exists between a tyrosine kinase receptor-mediated and a heterotrimeric G protein-coupled receptor-mediated signal transduction pathways. In the present study, we have studied effects of EGF receptor activation on activities of inhibitory G protein (Gi). We show that the amounts of Gi/Go ADP-ribosylated by islet-activating protein (IAP) increased by 30-40% in the membranes of Rat 1 fibroblast cells pretreated with EGF compared with those without pretreatment. When an effect of lysophosphatidic acid (LPA) stimulation on an adenylate cyclase activity was examined, LPA partly attenuated forskolin-stimulated adenylate cyclase activity via Gi because IAP pretreatment blocked the inhibitory effect of LPA. Pretreatment with EGF reduced the ability of LPA to inhibit the forskolin-stimulated adenylate cyclase activity, while the pretreatment did not have any effects on the forskolin-stimulated activity. Thus, the EGF receptor-mediated signal appears to cause the impairment of Gi function in Rat 1 fibroblast cells.     

Temperature-dependent stimulation and inhibition of adenylate cyclase by Aplysia bag cell peptides

The bag cell peptides (alpha-, beta-, and gamma-BCP) are secreted by the neuroendocrine bag cells of Aplysia, and provide feedback modulation of bag cell excitability and cAMP levels. We report here that if 200-500 mM NaCl is included in the assay buffer, the BCPs alter adenylate cyclase activity in a manner consistent with their effects on cAMP levels in intact bag cells. Specifically, beta-BCP and the related peptide A from the atrial gland stimulate the enzyme, while the effects of alpha-BCP(1-7) and gamma-BCP are temperature-dependent, stimulating at 30 degrees C and inhibiting at 15 degrees C. Both stimulation and inhibition require GTP, suggesting mediation by Gs and Gi. The ionic requirements of stimulation and inhibition differ: Cl- is necessary to support stimulation, but not inhibition. Moreover, pertussis toxin blocks inhibition, but does not affect stimulation. These results suggest that the temperature-sensitive mechanism lies upstream from the G-proteins in the signal transduction pathway.