Structural Basis of RNA Recognition by Mycobacterium tuberculosis MazF Homologues

The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.

Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.

In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.

Application and use of isothermal calorimetry in pharmaceutical development

There are many steps involved in developing a drug candidate into a formulated medicine and many involve analysis of chemical interaction or physical change. Calorimetry is particularly suited to such analyses as it offers the capacity to observe and quantify both chemical and physical changes in virtually any sample. Differential scanning calorimetry (DSC) is ubiquitous in pharmaceutical development, but the related technique of isothermal calorimetry (IC) is complementary and can be used to investigate a range of processes not amenable to analysis by DSC. Typically, IC is used for longer-term stability indicating or excipient compatibility assays because both the temperature and relative humidity (RH) in the sample ampoule can be controlled. However, instrument design and configuration, such as titration, gas perfusion or ampoule-breaking (solution) calorimetry, allow quantification of more specific values, such as binding enthalpies, heats of solution and quantification of amorphous content. As ever, instrument selection, experiment design and sample preparation are critical to ensuring the relevance of any data recorded. This article reviews the use of isothermal, titration, gas-perfusion and solution calorimetry in the context of pharmaceutical development, with a focus on instrument and experimental design factors, highlighted with examples from the recent literature. © 2011 Elsevier B.V.

Interaction between poly(ethylene glycol) and C12E8 investigated by dynamic light scattering, time-resolved fluorescence quenching, and calorimetry

Dynamic light scattering (DLS), time-resolved fluorescence quenching (TRFQ), and isothermal titration microcalorimetry have been used to show that, in dilute solution, low molecular weight poly(ethylene glycol) (PEG, M-w = 12 kDa) interacts with the nonionic surfactant octaethylene glycol n-dodecyl monoether, C12E8, to form a complex. Whereas the relaxation time distributions for the binary C12E8/water and PEG/water systems are unimodal, in the ternary mixtures they may be either uni- or bimodal depending on the relative concentrations of the components. At low concentrations of PEG or surfactant, the components of the relaxation time distribution are unresolvable, but the distribution becomes bimodal at higher concentrations of either polymer or surfactant. For the ternary system in excess surfactant, we ascribe, on the basis of the changes in apparent hydrodynamic radii and the scattered intensities, the fast mode to a single micelle, the surface of which is associated with the polymer and the slow mode to a similar complex but containing two or three micelles per PEG chain. Titration microcalorimetry results show that the interaction between C12E8, and PEG is exothermic and about 1 kJ mol(-1) at concentrations higher than the CMC of C12E8. The aggregation number, obtained by TRFQ, is roughly constant when either the PEG or the C12E8 concentration is increased at a given concentration of the second component, owing to the increasing amount of surfactant micelles inside the complex.

Ciclo reprodutivo de machos de Dendropsophus minutus (Anura, Hylidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Thermodynamic analysis of ferrous ion binding to Escherichia coli ferritin EcFtnA

Iron oxidation in the bacterial ferritin EcFtnA from Escherichia coli shows marked differences from its homologue human H-chain ferritin (HuHF). While the amino acid residues that constitute the dinuclear center in these proteins are highly conserved, EcFtnA has a third iron-binding site (C site) in close proximity to the dinuclear center that is seemingly responsible for these differences. Here, we describe the first thermodynamic study of Fe2+ binding to EcFtnA and its variants to determine the location of the primary ferrous ion-binding sites on the protein and to better understand the role of the third C site in iron binding. Isothermal titration calorimetric analyses of the wild-type protein reveal the presence of two main classes of binding sites in the pH range of 6.5-7.5, ascribed to Fe2+ binding, first at the A and then the B sites. Site-directed mutagenesis of ligands in the A, B, or C sites affects the apparent Fe2+-binding stoichiometries at the unaltered sites. The data imply some degree of inter- and intrasubunit negative cooperative interaction between sites. Unlike HuHF where only the A site initially binds Fe2+, both A and B sites in EcFtnA bind Fe2+, implying a role for the C site in influencing the binding of Fe2+ at the B site of the di-iron center of EcFtnA. The ITC equations describing a binding model for three classes of independent binding sites are reported here for the first time.

Observing the Solubilization of Lipid Bilayers by Detergents with Optical Microscopy of GUVs

The solubilization of lipid bilayers by detergents was studied with optical microscopy of giant unilamellar vesicles (GUVs) composed of palmitoyl oleoyl phoshatidylcholine (POPC). A solution of the detergents Triton X-100 (TX-100) and sodium dodecyl sulfate (SDS) was injected with a micropipette close to single GUVs. The solubilization process was observed with phase contrast and fluorescence microscopy and found to be dependent on the detergent nature. In the presence of TX-100, GUVs initially showed an increase in their surface area, due to insertion of TX-100 with rapid equilibration between the two leaflets of the bilayer. Then, above a solubility threshold, several holes opened, rendering the bilayer a lace fabric appearance, and the bilayer gradually vanished. On the other hand, injection of SDS caused initially an increase in the membrane spontaneous curvature, which is mainly associated with incorporation of SDS in the outer layer only. This created a stress in the membrane, which caused either opening of transient macropores with substantial decrease in vesicle size or complete vesicle bursting. In another experimental setup, the extent of solubilization/destruction of a collection of GUVs was measured as a function of either TX-100 or SDS concentration.

Deactivation of Ferrylmyoglobin by Vanillin as Affected by Vanillin Binding to beta-Lactoglobulin

Vanillin was found to be efficient as a deactivator of ferrylmyoglobin with a second-order rate constant of k(2) = S7 +/- 1 L mol(-1) s(-1) for reduction to metmyoglobin with Delta H(double dagger) = 58.3 +/- 0.3 kJ mol(-1) and Delta S(double dagger) = -14 +/- 1 J mol(-1) K(-1) in aqueous pH 7.4 solution at 25 degrees C. Binding to beta-lactoglobulin (AG) was found to affect the reactivity of vanillin at 25 degrees C only slightly to k(2) = 48 +/- 2 L mol(-1) s(-1) (Delta H(double dagger) = 68.4 +/- 0.4 kJ mol(-1) and Delta S(double dagger) = 17 +/- 1 J mol(-1) K(-1)) for deactivation of ferrylmyoglobin. Binding of vanillin to beta LG was found to have a binding stoichiometry vanillin/beta LG > 10 with K(A) = 6 x 10(2) L mol(-1) and an apparent total Delta H degrees of approximately -38 kJ mol(-1) and Delta S degrees = -S5.4 +/- 4J mol(-1) K(-1) at 25 degrees C and Delta C(p), (obs) = -1.02 kJ mol(-1) K(-1) indicative of increasing ordering in the complex, as determined by isothermal titration microcalorimetry. From tryptophan fluorescence quenching for beta LG by vanillin, approximately one vanillin was found to bind to each beta LG far stronger with K(A) = 5 x 10(4) L, mol(-1) and a Delta H degrees = 10.2 kJ mol(-1) and Delta S degrees = 55J mol(-1) K(-1) at 25 degrees C. The kinetic entropy/enthalpy compensation effect seen for vanillin reactivity by binding to beta LG is concluded to relate to the weakly bound vanillin oriented through hydrogen bonds on the beta LG surface with the phenolic group pointing toward the solvent, in effect making both Delta H(double dagger) and Delta S(double dagger) more positive. The more strongly bound vanillin capable of tryptophan quenching in the fiLG calyx seems less or nonreactive.

Binding of the cellulose-binding domain of exoglucanase Cex from Cellulomonas fimi to insoluble microcrystalline cellulose is entropically driven.

Isothermal titration microcalorimetry is combined with solution-depletion isotherm data to analyze the thermodynamics of binding of the cellulose-binding domain (CBD) from the beta-1,4-(exo)glucanase Cex of Cellulomonas fimi to insoluble bacterial microcrystalline cellulose. Analysis of isothermal titration microcalorimetry data against two putative binding models indicates that the bacterial microcrystalline cellulose surface presents two independent classes of binding sites, with the predominant high-affinity site being characterized by a Langmuir-type Ka of 6.3 (+/-1.4) x 10(7) M-1 and the low-affinity site by a Ka of 1.1 (+/-0.6) x 10(6) M-1. CBDCex binding to either site is exothermic, but is mainly driven by a large positive change in entropy. This differs from protein binding to soluble carbohydrates, which is usually driven by a relatively large exothermic standard enthalpy change for binding. Differential heat capacity changes are large and negative, indicating that sorbent and protein dehydration effects make a dominant contribution to the driving force for binding.

Inhibition of in vitro VEGF expression and choroidal neovascularization by synthetic dendrimer peptide mediated delivery of a sense oligonucleotide

Ocular neovascularisation is the leading cause of blindness in developed countries and the most potent angiogenic factor associated with neovascularisation is vascular endothelial growth factor (VEGF). We have previously described a sense oligonucleotide (ODN-1) that possesses anti-human and rat VEGF activity. This paper describes the synthesis of lipid-lysine dendrimers and their subsequent ability to delivery ODN-1 to its target and mediate a reduction in VEGF concentration both in vitro and in vivo. Positively charged dendrimers were used to deliver ODN-1 into the nucleus of cultured D407 cells. The effects on VEGF mRNA transcription and protein expression were analysed using RT-PCR and ELISA, respectively. The most effective dendrimers in vitro were further investigated in vivo using an animal model of choroidal neovascularisation (CNV). All dendrimer/ODN-1 complexes mediated in a significant reduction in VEGF expression during an initial 24 hr period (40-60%). Several complexes maintained this level of VEGF reduction during a subsequent, second 24 hr period, which indicated protection of ODN-1 from the effects of endogenous nucleases. In addition, the transfection efficiency of dendrimers that possessed 8 positive charges (chi = 81(.)51%) was significantly better (P = 0(.)0036) than those that possessed 4 positive charges (chi = 56(.)8%). RT-PCR revealed a correlation between levels of VEGF protein mRNA. These results indicated that the most effective structural combination was three branched chains of intermediate length with 8 positive charges such as that found for dendrimer 4. Dendrimer 4 and 7/ODN-1 complexes were subsequently chosen for in vivo analysis. Fluorescein angiography demonstrated that both dendrimers significantly (P < 0(.)0001) reduced the severity of laser mediated CNV for up to two months post-injection. This study demonstrated that lipophilic, charged dendrimer mediated delivery of ODN-1 resulted in the down-regulation of in vitro VEGF expression. In addition, in vivo delivery of ODN-1 by two of the dendrimers resulted in significant inhibition of CNV in an inducible rat model. Time course studies showed that the dendrimer/ODN-1 complexes remained active for up to two months indicating the dendrimer compounds provided protection against the effects of nucleases. (C) 2004 Elsevier Ltd. All rights reserved.

Effect of HMEC on the consolidation of cement pastes: Isothermal calorimetry versus oscillatory rheometry

Chemical admixtures increase the theological complexity of cement pastes owing to their chemical and physical interactions with particles, which affects cement hydration and agglomeration kinetics. Using oscillatory rheometry and isothermal calorimetry, this article shows that the cellulose ether HMEC (hydroxymethyl ethylcellulose), widely used as a viscosity modifying agent in self-compacting concretes and dry-set mortars, displayed a steric dispersant barrier effect during the first 2 h of hydration associated to a cement retarding nature, consequently reducing the setting speed. However, despite this stabilization effect, the polymer increased the cohesion strength when comparing cement particles with the same hydration degree. (C) 2009 Elsevier Ltd. All rights reserved.

A potential role for isothermal calorimetry in studies of the effects of thermodynamic non-ideality in enzyme-catalyzed reactions

Attention is drawn to the feasibility of using isothermal calorimetry for the characterization of enzyme reactions under conditions bearing greater relevance to the crowded biological environment, where kinetic parameters are likely to differ significantly from those obtained by classical enzyme kinetic studies in dilute solution. An outline of the application of isothermal calorimetry to the determination of enzyme kinetic parameters is followed by considerations of the nature and consequences of crowding effects in enzyme catalysis. Some of those effects of thermodynamic non-ideality are then illustrated by means of experimental results from calorimetric studies of the effect of molecular crowding on the kinetics of catalysis by rabbit muscle pyruvate kinase. This review concludes with a discussion of the potential of isothermal calorimetry for the experimental determination of kinetic parameters for enzymes either in biological environments or at least in media that should provide reasonable approximations of the crowded conditions encountered in vivo. Copyright (C) 2004 John Wiley Sons, Ltd.

Isothermal microcalorimetry: a novel method for real-time determination of antifungal susceptibility of Aspergillus species.

We evaluated microcalorimetry for real-time susceptibility testing of Aspergillus spp. based on growth-related heat production. The minimal heat inhibitory concentration (MHIC) for A. fumigatus ATCC 204305 was 1 mg/L for amphotericin B, 0.25 mg/L for voriconazole, 0.06 mg/L for posaconazole, 0.125 mg/L for caspofungin and 0.03 mg/L for anidulafungin. Agreement within two 2-fold dilutions between MHIC (determined by microcalorimetry) and MIC or MEC (determined by CLSI M38A) was 90% for amphotericin B, 100% for voriconazole, 90% for posaconazole and 70% for caspofungin. This proof-of-concept study demonstrated the potential of isothermal microcalorimetry for growth evaluation of Aspergillus spp. and real-time antifungal susceptibility testing.

Crystallization in poly(L-lactide)-b-poly(epsilon-caprolactone) double crystalline diblock copolymers: a study using X-ray scattering, differential scanning calorimetry, and polarized optical microscopy

The crystallization of well-defined poly(L-lactide)-b-poly(epsilon-caprolactone) diblock copolymers, PLLA-b-PCL, was investigated by time-resolved X-ray techniques, polarized optical microscopy (POM), and differential scanning calorimetry (DSC). Two compositions were studied that contained 44 and 60 wt % poly(L-lactide), PLLA (they are referred to as (L44C5614)-C-11 and (L60C409)-C-12, respectively, with the molecular weight of each block in kg/mol as superscript). The copolymers were found to be initially miscible in the melt according to small-angle X-ray scattering measurements (SAXS). Their thermal behavior was also indicative of samples whose crystallization proceeds from a mixed melt. Sequential isothermal crystallization from the melt at 100 degreesC (for 30 min) and then at 30 degreesC (for 15 min) was measured. At 100 degreesC only the PLLA block is capable of crystallization, and its crystallization kinetics was followed by both WAXS and DSC; comparable results were obtained that indicated an instantaneous nucleation with three-dimensional superstructures (Avrami index of approximately 3). The spherulitic nature of the superstructure was confirmed by POM. When the temperature was decreased to 30 degreesC, the PCL block was able to crystallize within the PLLA negative spherulites (with an Avrami index of 2, as opposed to 3 in homo-PCL), and its crystallization rate was much slower than an equivalent homo-PCL. Time-resolved SAXS experiments in (L60C409)-C-12 revealed an initial melt mixed morphology at 165 degreesC that upon cooling transformed into a transient microphase-separated lamellar structure prior to crystallization at 100 degreesC.

THERMAL BEHAVIOR STUDY AND DECOMPOSITION KINETICS OF SALBUTAMOL UNDER ISOTHERMAL AND NON-ISOTHERMAL CONDITIONS

The thermal decomposition of salbutamol (beta(2) - selective adrenoreceptor) was studied using differential scanning calorimetry (DSC) and thermogravimetry/derivative thermogravimetry (TG/DTG). It was observed that the commercial sample showed a different thermal profile than the standard sample caused by the presence of excipients. These compounds increase the thermal stability of the drug. Moreover, higher activation energy was calculated for the pharmaceutical sample, which was estimated by isothermal and non-isothermal methods for the first stage of the thermal decomposition process. For isothermal experiments the average values were E(act) = 130 kJ mol(-1) (for standard sample) and E(act) = 252 kJ mol(-1) (for pharmaceutical sample) in a dynamic nitrogen atmosphere (50 mL min(-1)). For non-isothermal method, activation energy was obtained from the plot of log heating rates vs. 1/T in dynamic air atmosphere (50 mL min(-1)). The calculated values were E(act) = 134 kJ mol(-1) (for standard sample) and E(act) (=) 139 kJ mol(-1) (for pharmaceutical sample).

A comparative study of the non-isothermal degradation of natural rubber from Mangabeira (Hancornia speciosa Gomes) and Seringueira (Hevea brasiliensis)

Due to the increasing search for alternative sources of natural rubber (NR) whose properties are similar to Hevea brasiliesis, several sources have been studied in the past few years. Among them, Mangabeira (Hancornia speciosa Gomes), which is native to Amazon rainforest and other regions of Brazil, has a potential as another viable rubber source. As a continuation of a series of comparative studies between Hancornia and Hevea (clone RRIM 600) these two species by our research team, their thermal behavior was analyzed by thermogravimetry (TG) using Flynn-Wall-Ozawa's approach in order to obtain kinetic parameters (reaction order, pre-exponential factor and activation energy) of the decomposition process. Results indicated that the thermal behavior of NR from Hancornia was comparable to Hevea with some differences observed as follows: reaction order for Hancornia was higher than for Hevea at the beginning of degradation and very close for temperatures over 350 A degrees C; activation energy and pre-exponential factor had the same trend, i.e., increased with increasing degree of conversion remaining almost constant between 20 and 70% and then increasing for higher degrees, although Hevea was slightly more thermally stable than Hancornia. These major influences in the degradation process in the early stage are attributed to differences in non-rubber constituents present in these two species.