Tobacco Upregulates P. gingivalis Fimbrial Proteins Which Induce TLR2 Hyposensitivity

Tobacco smokers are more susceptible to periodontitis than non-smokers but exhibit reduced signs of clinical inflammation. The underlying mechanisms are unknown. We have previously shown that cigarette smoke extract (CSE) represents an environmental stress to which P. gingivalis adapts by altering the expression of several virulence factors - including major and minor fimbrial antigens (FimA and Mfa1, respectively) and capsule - concomitant with a reduced pro-inflammatory potential of intact P. gingivalis.We hypothesized that CSE-regulation of capsule and fimbrial genes is reflected at the ultrastructural and functional levels, alters the nature of host-pathogen interactions, and contributes to the reduced pro- inflammatory potential of smoke exposed P. gingivalis. CSE induced ultrastructural alterations were determined by electron microscopy, confirmed by Western blot and physiological consequences studied in open-flow biofilms. Inflammatory profiling of specific CSE-dysregulated proteins, rFimA and rMfa1, was determined by quantifying cytokine induction in primary human innate and OBA-9 cells. CSE up-regulates P. gingivalis FimA at the protein level, suppresses the production of capsular polysaccharides at the ultrastructural level, and creates conditions that promote biofilm formation. We further show that while FimA is recognized by TLR2/6, it has only minimal inflammatory activity in several cell types. Furthermore, FimA stimulation chronically abrogates the pro-inflammatory response to subsequent TLR2 stimulation by other TLR-2-specific agonists (Pam3CSK4, FSL, Mfa1) in an IkappaBalpha- and IRAK-1-dependent manner.These studies provide some of the first information to explain, mechanistically, how tobacco smoke changes the P. gingivalis phenotype in a manner likely to promote P. gingivalis colonization and infection while simultaneously reducing the host response to this major mucosal pathogen

Protonation enthalpies for the active site residues of Trypanosoma brucei 6-phosphogluconate dehydrogenase

Inversions of “natural” ionization state of the ionisable residues in the active sites are frequently reported, but rarely it is known their energy cost. Here we report the energy cost of setting in the correct protonation state the active site of 6-phosphogluconate dehydrogenase (6PGDH) from T. brucei. 6PGDH is the third enzyme of the pentose phosphate pathway, catalyzing the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). 6PG binding to 6PGDH has been studied by ITC in the WT and mutants in the two residues mainly involved in catalysis, K185 and E192, whose protonation state invert upon the binding. From the pH dependence of the binding and activity it can be seen that binding of 6PG raises the pKa of E192 from 7.07 to 9.64, while it decreases the pKa of K185 from 9.9 to 7.17. E192 protonation gives a favourable enthalpy of ≈ -1.5 kcal/mole, while Lys185 deprotonation has an unfavourable enthalpy of ≈ +7.6 kcal/mole, with a net energy cost for the ionization exchange of 6.1 kcal/mole at the expense of the binding enthalpy. Since the ionization state of K185 and E192 in the enzyme-product complex is the same as in the free enzyme, thus the energy spent during 6PG binding will be used in driving the catalytic cycle. It is noteworthy that a theoretical model of 6PGDH catalysis predict that the non protonated K185 decreases the energy of the transition state of the dehydrogenation reaction by about 7.0 kcal/mole (1). (1) Wang J and Li S. Catalytic Mechanism of 6-Phosphogluconate Dehydrogenase: A Theoretical Investigation J. Phys. Chem. B 2006, 110, 7029-703

NADPH reduces oligomerization rate of a pre-existing dimer-tetramer equilibrium inTrypanosoma brucei 6-phosphogluconate dehydrogenase

6--Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). It not only gives NADPH and RU5P, but also depletes 6PG, whose accumulation induces cell senescence. It is a proposed drug target for African trypanosomiasis caused by T. brucei and for other microbial infections and cancer. We report here that the association of dimers to tetramers is an equilibrium present in the free enzyme, independently of the ligands. It has been shown by glutaraldehyde cross-linking, dynamic light scattering (DLS) and density gradient sedimentation. Both DLS and sedimentation indicate the enzyme size increases by increasing the enzyme concentration. In addition, gel filtration, density gradient sedimentation and isothermal titration calorimetry (ITC) reveal that the oligomerization rates are differently influenced by ligands. Indeed dynamic experiments where different oligomeric forms can be separated, show a strong NADPH shift of the enzyme versus the tetrameric form while NADP does not affect dimeric state of 6PGDH. Accordingly heat capacity change measured by ITC is different between NADP and NADPH binding (-92.56 against -520.35 cal/mol∙K) in agreement with a decreased solvent exposed surface area in tetramer. Tetramer is about 3-fold more active than dimer, indeed NADPH, the 6PGDH product and inhibitor, decreases interconversion rate while 6PG antagonizes NADPH effect. This is a further substrate way of promoting increased catalytic efficiency. The sheep liver 6PGDH, instead, by sedimentation studies appears always a dimer, the dimertetramer shift hence representing further drug exploitable potential

Protonation enthalpies for the active site residues of Trypanosoma brucei 6-phosphogluconate dehydrogenase

Inversions of “natural” ionization state of the ionisable residues in the active sites are frequently reported, but rarely it is known their energy cost. Here we report the energy cost of setting in the correct protonation state the active site of 6-phosphogluconate dehydrogenase (6PGDH) from T. brucei. 6PGDH is the third enzyme of the pentose phosphate pathway, catalyzing the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). 6PG binding to 6PGDH has been studied by ITC in the WT and mutants in the two residues mainly involved in catalysis, K185 and E192, whose protonation state invert upon the binding. From the pH dependence of the binding and activity it can be seen that binding of 6PG raises the pKa of E192 from 7.07 to 9.64, while it decreases the pKa of K185 from 9.9 to 7.17. E192 protonation gives a favourable enthalpy of ≈ -1.5 kcal/mole, while Lys185 deprotonation has an unfavourable enthalpy of ≈ +7.6 kcal/mole, with a net energy cost for the ionization exchange of 6.1 kcal/mole at the expense of the binding enthalpy. Since the ionization state of K185 and E192 in the enzyme-product complex is the same as in the free enzyme, thus the energy spent during 6PG binding will be used in driving the catalytic cycle. It is noteworthy that a theoretical model of 6PGDH catalysis predict that the non protonated K185 decreases the energy of the transition state of the dehydrogenation reaction by about 7.0 kcal/mole (1). (1) Wang J and Li S. Catalytic Mechanism of 6-Phosphogluconate Dehydrogenase: A Theoretical Investigation J. Phys. Chem. B 2006, 110, 7029-703

Evidence for dimer/tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway (PPP), is essential for biosyntheses and oxidative stress defence. It also has the function of depleting 6PG, whose accumulation induces cell senescence. 6PGDH is a proposed drug target for African trypanosomiasis caused by Trypanosoma brucei and for other microbial infections and cancer. Gel filtration, density gradient sedimentation, cross-linking and dynamic light scattering were used to assay the oligomerization state of T. brucei 6PGDH in the absence and presence of several ligands. The enzyme displays a dimer-tetramer equilibrium and NADPH (but not NADP) reduces the rate of approach to equilibrium, while 6PG is able to antagonize the NADPH effect. The different behaviour of the two forms of coenzyme appears to be related to the differences in Cp, with NADP binding Cp closer to what is expected of crystallographic structures, while NADPHCp is three times larger. The estimated dimer-tetramer association constant is 1.5 106 M-1, and the specific activity of the tetramer is about 3 fold higher than the specific activity of the dimer. Thus, cellular conditions promoting tetramer formation could allow an efficient clearing of 6PG. Experiments carried out on sheep liver 6PGDH indicate that tetramerization is a specificity of the parasite enzyme

Effects of heart disease on depression treatment: Results from the COMED study

10.1016/j.genhosppsych.2011.08.018General Hospital Psychiatry34124-34GHPS

NADPH reduces oligomerization rate of a pre-existing dimer-tetramer equilibrium in Trypanosoma brucei 6-phosphogluconate dehydrogenase

6-Phosphogluconate dehydrogenase (6PGDH), the third enzyme of the pentose phosphate pathway, catalyzes the NADP-dependent oxidative decarboxylation of 6-phosphogluconate (6PG) to ribulose-5-phosphate (RU5P). It not only gives NADPH and RU5P, but also depletes 6PG, whose accumulation induces cell senescence. It is a proposed drug target for African trypanosomiasis caused by T. brucei and for other microbial infections and cancer. We report here that the association of dimers to tetramers is an equilibrium present in the free enzyme, independently of the ligands. It has been shown by glutaraldehyde cross-linking, dynamic light scattering (DLS) and density gradient sedimentation. Both DLS and sedimentation indicate the enzyme size increases by increasing the enzyme concentration. In addition, gel filtration, density gradient sedimentation and isothermal titration calorimetry (ITC) reveal that the oligomerization rates are differently influenced by ligands. Indeed dynamic experiments where different oligomeric forms can be separated, show a strong NADPH shift of the enzyme versus the tetrameric form while NADP does not affect dimeric state of 6PGDH. Accordingly heat capacity change measured by ITC is different between NADP and NADPH binding (-92.56 against -520.35 cal/mol∙K) in agreement with a decreased solvent exposed surface area in tetramer. Tetramer is about 3-fold more active than dimer, indeed NADPH, the 6PGDH product and inhibitor, decreases interconversion rate while 6PG antagonizes NADPH effect. This is a further substrate way of promoting increased catalytic efficiency. The sheep liver 6PGDH, instead, by sedimentation studies appears always a dimer, the dimertetramer shift hence representing further drug exploitable potential

Energy cost for the proper ionization of active site residues in 6-phosphogluconate dehydrogenase from T. brucei

The catalytic mechanism of 6-phosphogluconate dehydrogenase requires the inversion of a Lys/Glu couple from its natural ionization state. The pKa of these residues in free and substrate bound enzymes has been determined measuring by ITC the proton release/uptake induced by substrate binding at different pH values. wt 6-phosphogluconate dehydrogenase from Trypanosoma brucei and two active site enzyme mutants, K185H and E192Q were investigated. Substrate binding was accompanied by proton release and was dependent on the ionization of a group with pKa 7.07 which was absent in the E192Q mutant. Kinetic data highlighted two pKa, 7.17 and 9.64, in the enzyme-substrate complex, the latter being absent in the E192Q mutant, suggesting that the substrate binding shifts Glu192 pKa from 7.07 to 9.64. A comparison of wt and E192Q mutant appears to show that the substrate binding shifts Lys185 pKa from 9.9 to 7.17. By comparing differences in proton release and the binding enthalpy of wt and mutants enzymes, the enthalpic cost of the change in the protonation state of Lys185 and Glu192 was estimated at ≈ 6.1 kcal/mole. The change in protonation state of Lys185 and Glu192 has little effect on Gibbs free energy, 240-325 cal/mol. However proton balance evidences the dissociation of other(s) group(s) that can be collectively described by a single pKa shift from 9.1 to 7.54. This further change in ionization state of the enzyme causes an increase of free energy with a total cost of 1.2-2.3 kcal/mol to set the enzyme into a catalytically competent form

Incidence, risk factors and outcome of barotrauma in mechanically ventilated patients.

OBJECTIVE: To determine the incidence, risk factors, and outcome of barotrauma in a cohort of mechanically ventilated patients where limited tidal volumes and airway pressures were used. DESIGN AND SETTING: Prospective cohort of 361 intensive care units from 20 countries. PATIENTS AND PARTICIPANTS: A total of 5183 patients mechanically ventilated for more than 12 h. MEASUREMENTS AND RESULTS: Baseline demographic data, primary indication for mechanical ventilation, daily ventilator settings, multiple-organ failure over the course of mechanical ventilation and outcome were collected. Barotrauma was present in 154 patients (2.9%). The incidence varied according to the reason for mechanical ventilation: 2.9% of patients with chronic obstructive pulmonary disease; 6.3% of patients with asthma; 10.0% of patients with chronic interstitial lung disease (ILD); 6.5% of patients with acute respiratory distress syndrome (ARDS); and 4.2% of patients with pneumonia. Patients with and without barotrauma did not differ in any ventilator parameter. Logistic regression analysis identified as factors independently associated with barotrauma: asthma [RR 2.58 (1.05-6.50)], ILD [RR 4.23 (95%CI 1.78-10.03)]; ARDS as primary reason for mechanical ventilation [RR 2.70 (95%CI 1.55-4.70)]; and ARDS as a complication during the course of mechanical ventilation [RR 2.53 (95%CI 1.40-4.57)]. Case-control analysis showed increased mortality in patients with barotrauma (51.4 vs 39.2%; p=0.04) and prolonged ICU stay. CONCLUSIONS: In a cohort of patients in whom airway pressures and tidal volume are limited, barotrauma is more likely in patients ventilated due to underlying lung disease (acute or chronic). Barotrauma was also associated with a significant increase in the ICU length of stay and mortality