Positive feedback and noise activate the stringent response regulator rel in mycobacteria

Phenotypic heterogeneity in an isogenic, microbial population enables a subset of the population to persist under stress. In mycobacteria, stresses like nutrient and oxygen deprivation activate the stress response pathway involving the two-component system MprAB and the sigma factor, SigE. SigE in turn activates the expression of the stringent response regulator, rel. The enzyme polyphosphate kinase 1 (PPK1) regulates this pathway by synthesizing polyphosphate required for the activation of MprB. The precise manner in which only a subpopulation of bacterial cells develops persistence, remains unknown. Rel is required for mycobacterial persistence. Here we show that the distribution of rel expression levels in a growing population of mycobacteria is bimodal with two distinct peaks corresponding to low (L) and high (H) expression states, and further establish that a positive feedback loop involving the mprAB operon along with stochastic gene expression are responsible for the phenotypic heterogeneity. Combining single cell analysis by flow cytometry with theoretical modeling, we observe that during growth, noise-driven transitions take a subpopulation of cells from the L to the H state within a "window of opportunity" in time preceding the stationary phase. It is these cells which adapt to nutrient depletion in the stationary phase via the stringent response. We find evidence of hysteresis in the expression of rel in response to changing concentrations of PPK1. Hysteresis promotes robustness in the maintenance of the induced state. Our results provide, for the first time, evidence that bistability and stochastic gene expression could be important for the development of "heterogeneity with an advantage" in mycobacteria and suggest strategies for tackling tuberculosis like targeting transitions from the low to the high rel expression state.     

Arginine therapy of transgenic-knockout sickle mice improves microvascular function by reducing non-nitric oxide vasodilators, hemolysis, and oxidative stress

In sickle cell disease, nitric oxide (NO) depletion by cell-free plasma hemoglobin and/or oxygen radicals is associated with arginine deficiency, impaired NO bioavailability, and chronic oxidative stress. In transgenic-knockout sickle (BERK) mice that express exclusively human alpha- and beta(S)-globins, reduced NO bioavailability is associated with induction of non-NO vasodilator enzyme, cyclooxygenase (COX)-2, and impaired NO-mediated vascular reactivity. We hypothesized that enhanced NO bioavailability in sickle mice will abate activity of non-NO vasodilators, improve vascular reactivity, decrease hemolysis, and reduce oxidative stress. Arginine treatment of BERK mice (5% arginine in mouse chow for 15 days) significantly reduced expression of non-NO vasodilators COX-2 and heme oxygenase-1. The decreased COX-2 expression resulted in reduced prostaglandin E(2) (PGE(2)) levels. The reduced expression of non-NO vasodilators was associated with significantly decreased arteriolar dilation and markedly improved NO-mediated vascular reactivity. Arginine markedly decreased hemolysis and oxidative stress and enhanced NO bioavailability. Importantly, arteriolar diameter response to a NO donor (sodium nitroprusside) was strongly correlated with hemolytic rate (and nitrotyrosine formation), suggesting that the improved microvascular function was a response to reduced hemolysis. These results provide a strong rationale for therapeutic use of arginine in sickle cell disease and other hemolytic diseases.     

Orally efficacious thrombin inhibitors with cyanofluorophenylacetamide as the P2 motif

2-Cyano-6-fluorophenylacetamide was explored as a novel P2 scaffold in the design of thrombin inhibitors. Optimization around this structural motif culminated in 14, which is a potent thrombin inhibitor (K_i = 1.2 nM) that exhibits robust efficacy in canine anticoagulation and thrombosis models upon oral administration.     

Kinetics and mechanism of the reduction of the molybdatopentaamminecobalt(III) ion by aqueous sulfite and aqueous potassium hexacyanoferrate(II)

A detailed investigation on the oxidation of aqueous sulfite and aqueous potassium hexacyanoferrate(II) by the title complex ion has been carried out using the stopped-flow technique over the ranges, 0.01≤[S(IV)]_T≤0.05 mol dm⁻³, 4.47≤pH≤5.12, and 24.9≤θ≤37.6 °C and at ionic strength 1.0 mol dm⁻³ (NaNO₃) for aqueous sulfite and 0.01≤[Fe(CN)₆ ⁴⁻]≤0.11 mol dm⁻³, 4.54≤pH≤5.63, and 25.0≤θ≤35.3 °C and at ionic strength 1.0 or 3.0 mol dm⁻³ (NaNO₃) for the hexacyanoferrate(II) ion. Both redox processes are dependent on pH and reductant concentration in a complex manner, that is, for the reaction with aqueous sulfite, k_obs={(k₁K₁K₂K₃+k₂K₁K₄[H⁺])[S(IV)]_T]/([H⁺]²+K₁[H⁺]+K₁K₂) and for the hexacyanoferrate(II) ion, k_obs={(k₁K₃K₄K₅+k₂K₃K₆[H⁺])[Fe(CN)₆ ⁴⁻]_T)/([H⁺]²+K₃[H⁺]+K₃K₄). At 25.0 °C, the value of k₁′ (the composite of k₁K₃) is 0.77±0.07 mol⁻¹ dm³ s⁻¹, while the value of k₂′ (the composite of k₂K₄) is (3.78±0.17)×10⁻² mol⁻¹ dm³ s⁻¹ for aqueous sulfite. For the hexacyanoferrate(II) ion, k₁′ (the composite of k₁K₅) is 1.13±0.01 mol⁻¹ dm³ s⁻¹, while the value of k₂′ (the composite of k₂K₆) is 2.36±0.05 mol⁻¹ dm³ s⁻¹ at 25.0 °C. In both cases there was reduction of the cobalt(III) centre to cobalt(II), but there was no reduction of the molybdenum(VI) centre. k₂₂, the self-exchange rate constant, for aqueous sulfite (as SO₃ ²⁻) was calculated to be 5.37×10⁻¹² mol⁻¹ dm³ s⁻¹, while for Fe(CN)₆ ⁴⁻, it was calculated to be 1.10×10⁹ mol⁻¹ dm³ s⁻¹ from the Marcus equations.     

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I. Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Aspergillus niger

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Aspergillus niger was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34 mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the ortho-position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.     

Sialylation of lacto-N-neohexaosylceramide by sialyltransferase from embryonic chicken muscle

A sialyltransferase which catalyzes the in vitro biosynthesis of N-acetylneuraminosyllacto-N-neohexaosylceramide from lacto-N-neohexaosylceramide and CMP-NeuAc has been examined in embryonic chicken breast muscle. The maximum enzyme activity was observed in 11-12-day-old embryos. The enzyme has optimum activity at pH 6.8 in the presence of Triton CF-54 and Mg2+. The apparent Km values for lacto-N-neohexaosylceramide and CMP-NeuAc were 0.9 and 0.67 mM, respectively. The enzymic product was characterized by TLC, neuraminidase hydrolysis and permethylation analysis. The structure was identical to authentic N-acetylneuraminosyllacto-N-neohexaosylceramide from chicken muscle. In addition, a disialo derivative has been detected that constitutes 15% of the total radioactivity incorporated. The two sialic acids connected by sialosyl-sialosyl linkage were attached to the terminal galactose residue. To our knowledge, this is the first report of biosynthesis of this disialo compound.     

Cryogenic stabilization of myoglobin photoproducts

The low frequency resonance Raman spectra of photodissociated carbon monoxymyoglobin at cryogenic temperatures (4-77 K) differ from those of deoxymyoglobin. Intensity differences occur in several low frequency porphyrin modes, and intensity and frequency differences occur in the iron-histidine stretching mode. This mode appears at about 225 cm-1 in deoxymyoglobin. At the lowest temperature studied, approximately 4 K, the frequency of the iron-histidine stretching mode in the photoproduct is approximately 233 cm-1, and the intensity is very low. When the temperature of the photoproduct is increased, the intensity of the mode increases, but its frequency is unchanged. The differences between the photoproduct and the deoxy preparation persist to 77 K, the highest temperature studied, and are independent of whether samples are frozen in phosphate buffer or a 50:50 ethylene glycol/phosphate buffer mixture. It is proposed that the frequency of the iron-histidine stretching mode is governed by the tilt angle of the histidine with respect to the normal to the heme plane, and the intensity of the mode is governed by the overlap between the sigma orbital of the iron-histidine bond and the pi orbital of the porphyrin macrocycle. This model can account for differences between the resonance Raman spectra of the photoproduct and the deoxy preparations of both hemoglobin and myoglobin. Furthermore, by considering the F-helix motions in going from 6-coordinate to 5-coordinate hemoglobin and myoglobin, the heme relaxation of these proteins at room temperature with 10-ns pulses can be explained. Based on the findings reported here, low temperature relaxation pathways for both hemoglobin and myoglobin are proposed.