The role of hydration on the mechanism of allosteric regulation: In situ measurements of the oxygen-linked kinetics of water binding to hemoglobin

We report here the first direct measurements of changes in protein hydration triggered by a functional binding. This task is achieved by weighing hemoglobin (Hb) and myoglobin films exposed to an atmosphere of 98%, relative humidity during oxygenation. The binding of the first oxygen molecules to Hb tetramer triggers a change in protein conformation, which increases binding affinity to the remaining empty sites giving rise to the appearance of cooperative phenomena. Although crystallographic data have evidenced that this structural change increases the protein water-accessible surface area, isobaric osmotic stress experiments in aqueous cosolutions have shown that water binding is linked to Hb oxygenation. Now we show that the differential hydration between fully oxygenated and fully deoxygenated states of these proteins, determined by weighing protein films with a quartz crystal microbalance, agree with the ones determined by osmotic stress in aqueous cosolutions, from the linkage between protein oxygen affinity and water activity. The agreements prove that the changes in water activity brought about by adding osmolytes to the buffer solution shift biochemical equilibrium in proportion to the number of water molecules associated with the reaction. The concomitant kinetics of oxygen and of water binding to Hb have been also determined. The data show that the binding of water molecules to the extra protein surface exposed on the transition from the low-affinity T to the high-affinity R conformations of hemoglobin is the rate-limiting step of Hb cooperative reaction. This evidences that water binding is a crucial step on the allosteric mechanism regulating cooperative interactions, and suggests the possibility that environmental water activity might be engaged in the kinetic control of some important reactions in vivo.

Purification, crystallization and preliminary X-ray analysis of haemoglobin I from the armoured catfish Liposarcus anisitsi

Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 Angstrom resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique.

Water regulation of actinomycin-D binding to DNA: the interplay among drug affinity, DNA long-range conformation, and hydration

Actiaomycin-D (actD) binds to natural DNA at two different classes of binding sites, weak and strong. The affinity for these sites is highly dependent on DNA se(sequence and solution conditions, and the interaction appears to be purely entropic driven Although the entropic character of this reaction has been attributed to the release of water molecules upon drug to DNA complex formation, the mechanism by which hydration regulates actD binding and discrimination between different classes of binding sites on natural DNA is still unknown. In this work, we investigate the role of hydration on this reaction using the osmotic stress method. We skew that the decrease of solution water activity, due to the addition of sucrose, glycerol ethylene glycol, and betaine, favors drug binding to the strong binding sites on DNA by increasing both the apparent binding affinity Delta G, and the number of DNA base pairs apparently occupied by the bound drug n(bp/actD). These binding parameters vary linearly with the logarithm of the molar fraction of water in solution log(X-w), which indicates the contribution of water binding to the energetic of the reaction. It is demonstrated that the hydration change measured upon binding increases proportionally to the apparent size of the binding site n(bp/uctD). This indicates that n(bp/actD) measured from the Scatchard plod is a measure of the size of the DNA molecule changing conformation due to ligand binding. We also find that the contribution of DNA deformation, gauged by n(bp/act) to the total free energy of binding Delta G, is given by Delta G = Delta G(local) + n(bp/actD) x delta G(DNA), where Delta G(local), = -8020 +/- 51 cal/mol of actD bound and delta G(DNa) = -24.1 +/- 1.7cal/mol of base pair at 25 degrees C. We interpret Delta G(local), as the energetic contribution due to the direct interactions of actD with the actual tetranucleotide binding site, and it n(bp/actB) X delta G(DNA) as that due to change inconformation, induced by binding, of it n(bp/actD) DNA base pairs flanking the local site. This interpretation is supported by the agreement found between the value of delta G(DNA) and the torsional free energy change measured independently. We conclude suggesting an allosteric model for ligand binding to DNA, such that the increase in binding affinity is achieved by increasing the relaxation of the unfavorable free energy of binding storage at the local site through a larger number of DNA base pairs. The new aspect on this model is that the size of the complex is not fixed but determined by solutions conditions, such as water activity, which modulate the energetic barrier to change helix conformation. These results may suggest that long-range allosteric transitions of duplex DNA are involved in the inhibition of RNA synthesis by actD, and more generally, in the regulation of transcription. (C) 2000 John Wiley & Sons, Inc.

Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A(0) fully stripped of any anions

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O-2-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 Angstrom resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

Transposable elements in Coffea (Gentianales : Rubiacea) transcripts and their role in the origin of protein diversity in flowering plants

Transposable elements are major components of plant genomes and they influence their evolution, acting as recombination hot spots, acquiring specific cell functions or becoming part of protein-coding regions. The latter is the subject of the present analysis. This study is a report on the annotation of transposable elements (TEs) in expressed sequences of Coffea arabica, Coffea canephora and Coffea racemosa, showing the occurrence of 383 ESTs and 142 unigenes with TE fragments in these three Coffea species. Based on selected unigenes, it was possible to suggest 26 putative proteins with TE-cassette insertions, demonstrating a likely contribution to protein variability. The genes for two of those proteins, the fertility restorer (FR) and the pyrophosphate-dependent phosphofructokinase (PPi-PFKs) genes, were selected for evaluating the impact of TE-cassettes on host gene evolution of other plant genomes (Arabidopsis thaliana, Oryza sativa and populus trichocarpa). This survey allowed identifying a FR gene in O. sativa harboring multiple insertions of LTR retrotransposons that originated new exons, which however does not necessarily mean a case of molecular domestication. A possible transduction event of a fragment of the PPi-PFK beta-subunit gene mediated by Helitron ATREPX1 in Arabidopsis thaliana was also highlighted.

Repetition of Suicide Attempts Data from Emergency Care Settings in Five Culturally Different Low- and Middle-Income Countries Participating in the WHO SUPRE-MISS Study

Background: Attempted suicide is a strong risk factor for subsequent suicidal behaviors. Innovative strategies to deal with people who have attempted suicide are needed, particularly in resource-poor settings. Aims: To evaluate a brief educational intervention and periodic follow-up contacts (BIC) for suicide attempters in five culturally different sites (Campinas, Brazil; Chennai, India; Colombo, Sri Lanka; Karaj, Islamic Republic of Iran; and Yuncheng, People's Republic of China) as part of the WHO Multisite Intervention Study on Suicidal Behaviors (SUPRE-MISS). Methods: Among the 1,867 suicide attempters enrolled in the emergency departments of the participating sites, 922 (49.4%) were randomly assigned to a brief intervention and contact (BIC) group and 945 (50.6%) to a treatment as usual (TAU) group. Repeated suicide attempts over the 18 months following the index attempt - the secondary outcome measure presented in this paper - were identified by follow-up calls or visits. Subsequent completed suicide - the primary outcome measure has been reported in a previous paper. Results: Overall, the proportion of subjects with repeated suicide attempts was similar in the BIC and TAU groups (7.6% vs. 7.5%, chi(2) = 0.013; p = .909), but there were differences in rates across the five sites. Conclusions: This study from five low-and middle-income countries does not confirm the effectiveness of brief educational intervention and follow-up contacts for suicide attempters in reducing subsequent repetition of suicide attempts up to 18 months after discharge from emergency departments.

Intrageneric Karyotypic Variation in Pseudopaludicola (Anura: Leiuperidae) and Its Taxonomic Relatedness

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Hydration-dependent conformational states of hemoglobin. Equilibrium and kinetic behavior

The equilibrium and kinetics of methemoglobin conversion to hemichrome induced by dehydration were investigated by visible absorption spectroscopy. Below about 0.20 g water per g hemoglobin only hemichrome was present in the sample; above this value, an increasing proportion of methemoglobin appeared with the increase in hydration. The transition between the two derivatives showed a time-dependent biphasic behavior and was observed to be reversible. The rates obtained for the transition of methemoglobin to hemichrome were 0.31 and 1.93 min-1 and for hemichrome to methemoglobin 0.05 and 0.47 min-1. We suggest that hemichrome is a reversible conformational state of hemoglobin and that the two rates observed for the transition between the two derivatives reflect the α- and β-chains of hemoglobin.

OLEO ESSENCIAL EM ALGUMAS ESPECIES DE EUCALYPTUS

In this work we used leaves of four species of Eucalyptus - E. bicostata Maiden, E. deanei Maiden, E. dunnii Maiden and E. viminalis Labill, grown in Colombo, Parana State, Brazil. Preliminary studies were done, in the following sequence: botanic identification, olfactory research, extraction and purification of the essential oil of each species. The physical and chemical tests done were the following: relative density, refractive index, optical rotation, solubility, coagulation point and cineol percentage. The essential oils analysed through gaseous chromatography, presented the following cineol percentage: E. bicostata Maiden - 26.92%, E. deanei Maiden - 70.21%, E. dunnii Maiden - 53.16% and E. viminalis Labill - 23.05%.

Middle Proterozoic vein-hosted gold deposits in the Pontes e Lacerda region, southwestern Amazonian craton, Brazil

Structural, geochemical, and isotope studies were carried out on the gold deposits of the Pontes e Lacerda region (Mato Grosso state, Brazil), where rocks of the Aguapei and Rondoniano mobile belts (southwestern Amazonian craton) occur. The orebodies are hosted in metavolcanic, gneiss-granite, quartzite, tonalite, and granite units. Tectonics involve oblique overthrusting (from northeast to southwest), which led to the formation of recumbent folds and thrusts (pathways for the mineralizing fluids), upright folds, and faults with dominant strike-slip component. These unconformities represent potential sites for mineralization. During geological mapping, it was observed that the orebodies consist of quartz, pyrite, and gold, and that the hydrothermal alteration zone contains quartz, sericite, pyrite (altered to limonite), and magnetite (altered to hematite). Chalcopyrite, galena, and sphalerite occur only in the Onça deposit. Chemical analysis of sulfides indicates high contents of Bi, Se, and Te in sulfides and gold, suggesting plutonic involvement in the origin of hydrothermal solutions. K-Ar dating of hydrothermal sericites from gold veins yielded ages in the range from 960 to 840 Ma, which may indicate the age of original crystallization of sericite. Pb-Pb dating in galenas yielded model ages in the range from 1000 to 800 Ma for the Onça deposit, which is in agreement with K-Ar ages. Pb-isotopic ratios indicate high U/Pb and low Th/Pb for the upper-crustal Pb source before incorporation in galena crystals. The Pontes e Lacerda gold deposits yielded ages correlated to the Aguapei event and probably were formed during a Proterozoic contractional tectonic period in the southwestern part of the Amazon craton, which may characterize an important metallogenic epoch in the Pontes e Lacerda region.

Crystallization and X-ray diffraction data analysis of oxyhemoglobin-I from the catfish Liposarcus anisitsi (pisces)

Hemoglobin remains, despite the enormous amount of research involving this molecule, as a prototype for allosteric models and new conformations. Functional studies carried out on Hemoglobin-I from the South-American Catfish Liposarcus anisitsi [1] suggest the existence of conformational states beyond those already described for human hemoglobin, which could be confirmed crystallographically. The present work represents the initial steps towards that goal.

Histological study of hamster buccal mucosa following topical application of DMBA and exposition to low power 337 nm laser light

The aim of the present work is to analyze the histological changes on hamster buccal mucosa caused by the topical use of 7,12-dimethylbenzanthracene (DMBA) and exposition to a 220 μJ/pulse nitrogen laser light (@ 337 nm) at an average power of 2,3 mW. Twenty-one hamsters divided into two experimental groups were treated six times with DMBA. One hamster was kept as control. Group I was composed by ten hamsters and was submitted only to DMBA. Group II, also with ten hamsters, received the same treatment as group I and was exposed to the laser radiation. The time duration of each irradiation section was 10 seconds. All the treatment happened in alternated days. The histological analysis took place twice, after the end of the treatment and after sixty days. Both experimental groups presented dilatation of vessels, thickening of the epithelial tissue and the presence of inflammatory infiltrates. The preliminary results indicates that in group II the number of dilated vessels and its new area are much more significant than in group I.

GSTT1, GSTM1 and CYP2E1 genetic polymorphisms in gastric cancer and chronic gastritis in a Brazilian population

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)