Circular proteins - no end in sight

Circular proteins are a recently discovered phenomenon. They presumably evolved to confer advantages over ancestral linear proteins while maintaining the intrinsic biological functions of those proteins. In general, these advantages include a reduced sensitivity to proteolytic cleavage and enhanced stability. In one remarkable family of circular proteins, the cyclotides, the cyclic backbone is additionally braced by a knotted arrangement of disulfide bonds that confers additional stability and topological complexity upon the family. This article describes the discovery, structure, function and biosynthesis of the currently known circular proteins. The discovery of naturally occurring circular proteins in the past few years has been complemented by new chemical and biochemical methods to make synthetic circular proteins; these are also briefly described.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

SAM (dependent) I AM: the S-adenosylmethionine-dependent methyltransferase fold

The S-adenosylmethionine-dependent methyltransferase enzymes share little sequence identity, but incorporate a highly conserved structural fold. Surprisingly, residues that bind the common cofactor are poorly conserved, although the binding site is localised to the same region of the fold. The substrate-binding region of the fold varies enormously. Over the past two years, there has been a significant increase in the number of structures that are known to incorporate this fold, including several uncharacterised proteins and two proteins that lack methyltransferase activity.

Mechanisms for the inhibition of amiloride-sensitive Na+ absorption by extracellular nucleotides in mouse trachea

Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V-te) and amiloride-sensitive short-circuit current (Isc-Amil) in mouse trachea. Exposure to apical ATP or UTP (each 100 mumol/l) caused a large initial increase in lumen negative V-te and I-sc corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of Isc-Amil that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 mumol/l) or the Ca2+ ionophore ionomycin (1 mumol/l). Removal of extracellular Cl- or exposure to 200 muM DIDS reduced UTP-mediated inhibition of Isc-Amil Substantially. The phospholipase inhibitor U73122 (10 mumol/l) and pertussis toxin (PTX 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of Isc-Amil. Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.

Antibiotic desensitization in adults with cystic fibrosis

Objective: Allergic reactions to antibiotics occur in up to 30% of patients with cystic fibrosis (CF). Repeated antibiotic exposure and immune hyper-responsiveness increase the risk of allergic reactions and may limit antibiotic choice. Desensitization may allow the successful administration of an antibiotic despite previous allergy. We aimed to determine the success of antibiotic desensitization in patients with CF in an adult CF unit over a 7-year period. Methodology: A retrospective medical record review was performed on the 19 patients who had undergone antibiotic desensitization procedures. Data collected included drug allergy and intolerance profiles, nature of allergies, and the outcome of desensitization procedures. Desensitization procedures were performed in a ward setting according to published methods. Results: Nineteen patients (13 females) reported 62 drug allergies with a mean of 3.3 per patient. Of the 71 desensitization procedures undergone by this group, 54 (76%) were successful. Fifteen of the 19 patients were allergic to two or more beta-lactam antibiotics. Over half of the patients were desensitized to more than one antibiotic. Nine different antibiotics were used in 31 different patient/drug combinations. A successful outcome was achieved in 18/31 (58%) combinations, with three requiring treatment for mild allergic reactions. Allergic reactions caused drug cessation in a total of 19 patient/drug combinations (three after initial successful desensitization and full courses of antibiotics). Over 50% of these reactions occurred on day 1. Desensitization failures were more common in patients with well-documented allergic reactions to a specific drug. Conclusion: This study demonstrates that multiple antibiotic allergies are common in adults with CE Cross-reactivity between beta-lactam antibiotics may limit antibiotic choice for the treatment of pulmonary exacerbations. Antibiotic desensitization allows safe and successful treatment in the ward setting of many patients with previous allergies to an antibiotic. In many patients symptoms of allergy still occur and result in cessation of the antibiotics. Use of corticosteroids and antihistamines may improve the success rate of desensitization procedures.

Functionalized macrocyclic compounds: potential sensors of small molecules and ions

The potential applications of macrocycles in chemistry and at its interfaces with biology and physics continue to emerge, one of which is as receptors for small molecules and ions. This review illustrates these applications with examples from the last ten years employing complexation as the binding mechanism; some of the systems presented have already found real-world sensor applications. In any case, the challenges remain to design more selective and sensitive receptors for guests.

A eficácia e segurança da fisioterapia respiratória no tratamento da bronquiolite aguda em crianças até 2 anos de idade: revisão sistemática

Contexto: a bronquiolite aguda é a principal patologia a afectar a criança nos primeiros 2 anos de vida, a fisioterapia respiratória é uma intervenção terapêutica utilizada com a intenção de melhorar o curso desta doença mantendo-se a incerteza sobre a sua eficácia. Objectivo: determinar a eficácia e segurança da fisioterapia respiratória em crianças com menos de 2 anos com bronquiolite aguda. Fontes de Informação: Medline (1966 a Agosto 2010), EMBASE(1990 a Agosto 2010), Pedro e Lilacs (1982 a Agosto 2010). Outra fonte de informação incluiu a bibliografia dos estudos obtidos. Selecção de estudos: estudos experimentais comparando a fisioterapia respiratória com cuidados habituais, em crianças com menos de 2 anos e bronquiolite aguda, em ventilação espontânea, em qualquer contexto. Estudos pré-experimentais ou observacionais com os mesmos participantes e intervenções foram admitidos complementarmente aos experimentais. Extracção de dados e análise: um investigador extraiu os dados dos artigos obtidos e avaliou o risco de viés. A eficácia e segurança da fisioterapia respiratória foram determinadas pelos seguintes outcomes: duração do internamento hospitalar ou do evento, variação de scores de severidade clínica, saturação periférica e suplementação de oxigénio, recidivas, recurso a antibióticos e efeitos deletérios ou deterioração clínica reportada. Síntese de dados: 6 estudos experimentais foram admitidos. As suas amostras provinham de criança internadas em hospital. As técnicas de fisioterapia respiratória foram comparadas com cuidados habituais. Nenhum estudo evidenciou melhoria dos outcomes de interesse na comparação entre grupos, excepto avaliações de curta duração da saturação periférica de oxigénio e scores de severidade clínica. 1 estudo reportou uma percentagem significativamente maior no grupo submetido a fisioterapia respiratória de crianças que vomitaram, tiveram uma desestabilização respiratória transitória, e na percepção de stress da criança pelos cuidadores. São relatadas ainda fracturas costais a causa de fisioterapia respiratória. Limitações: o risco de viés era alto em 2 estudos, baixo num estudo e indeterminado nos restantes. Conclusões: aparentemente a fisioterapia respiratória não é eficaz e pode produzir efeitos deletérios importantes, mas a evidência é pobre, carecendo de novos estudos.

Biochemical and structural characterization of β-lactamases tem-180 and tem-201

With the constant development of new antibiotics, selective pressure is a force to reckon when investigating antibiotic resistance. Although advantageous for medical treatments, it leads to increasing resistance. It is essential to use more potent and toxic antibiotics. Enzymes capable of hydrolyzing antibiotics are among the most common ways of resistance and TEM variants have been detected in several resistant isolates. Due to the rapid evolution of these variants, complex phenotypes have emerged and the need to understand their biological activity becomes crucial. To investigate the biochemical properties of TEM-180 and TEM-201 several computational methodologies have been used, allowing the comprehension of their structure and catalytic activity, which translates into their biological phenotype. In this work we intent to characterize the interface between these proteins and the several antibiotics used as ligands. We performed explicit solvent molecular dynamics (MD) simulations of these complexes and studied a variety of structural and energetic features. The interfacial residues show a distinct behavior when in complex with different antibiotics. Nevertheless, it was possible to identify some common Hot Spots among several complexes – Lys73, Tyr105 and Glu166. The structural changes that occur during the Molecular Dynamic (MD) simulation lead to the conclusion that these variants have an inherent capacity of adapting to the various antibiotics. This capability might be the reason why they can hydrolyze antibiotics that have not been described until now to be degraded by TEM variants. The results obtained with computational and experimental methodologies for the complex with Imipenem have shown that in order to this type of enzymes be able to acylate the antibiotics, they need to be capable to protect the ligand from water molecules.

From antimicrobial agents to health promotion

A emergência de multiresistência apresentada por microrganismos é um dos grandes desafios que enfrentam actualmente os profissionais de Saúde e a população em geral. Os factores que contribuem para o desenvolvimento de resistência a antibióticos na comunidade podem ser categorizados como comportamentais ou ambientais/políticas. O objectivo deste trabalho foi caracterizar a situação actual na visão dos Pais de alunos do pré-escolar e 1º ciclo. De modo a avaliar as necessidades de intervenção e as actividades a serem desenvolvidas, um instrumento para estudar os hábitos e comportamentos adoptados na utilização de antibióticos, foi adaptado, validado e aplicado numa amostra piloto.

Evaluation of pharmaceutical assistance in public primary care in Brasília, Brazil

OBJECTIVE: Pharmaceutical assistance is essential in health care and a right of citizens according to Brazilian law and drug policies. The study purpose was to evaluate aspects of pharmaceutical assistance in public primary health care. METHODS: A cross-sectional study using WHO drug indicators was carried out in Brasília in 2001. From a random sample of 15 out of 62 centers thirty exiting patients per center were interviewed. RESULTS: Only 18.7% of the patients fully understood the prescription, 56.3% could read it, 61.2% of the prescribed drugs were actually dispensed, and mean duration of pharmaceutical dispensing was 53.2 seconds. Each visit lasted on average 9.4 minutes. Of prescribed and non-dispensed drugs, 85.3% and 60.6% were on the local essential drug list (EDL) respectively. On average 83.2% of 40 essential drugs were in stock, and only two centers had a pharmacist in charge of the pharmacy. The mean number of drugs per prescription was 2.3, 85.3% of prescribed drugs were on the EDL, 73.2% were prescribed using the generic denomination, 26.4% included antibiotics and 7.5% were injectables. The most prescribed groups were: cardiovascular drugs (26.8%), anti-infective drugs (13.1%), analgesics (8.9%), anti-asthmatic drugs (5.8%), anti-diabetic drugs (5.3%), psychoactive drugs (3.7%), and combination drugs (2.7%). CONCLUSIONS: Essential drugs were only moderately available almost 30 years after the first Brazilian EDL was formulated. While physician use of essential drugs and generic names was fairly high, efficiency was impaired by the poor quality of pharmaceutical care, resulting in very low patient understanding and insufficient guarantee of supply, particularly for chronic diseases.

Preparation and characterization of beta-lactam antibiotic as Ionic liquids and salts

With the increase of bacterial resistance a large number of therapeutic strategies have been used to fight different kind of infections. In recent years ionic liquids (ILs) have been increasing the popularity and the number of applications. First ionic liquids were used mainly as solvent in organic synthesis, but now they are used in analytical chemistry, separation chemistry and material science among others. Additional to significant developments in their chemical properties and applications, ionic liquids are now bringing unexpected opportunities at the interface of chemistry with the life sciences Ionic liquids (ILs) are currently defined as salts that are composed solely of cations and anions which melt below 100ºC. Our goal in this work is to explore the dual activity of the ionic liquids, due to the presence of two different ions, an ion with bacterial activity as a beta-lactam antibiotic and different kinds of cations. In this work the anions of ILs and salts were derived from three different antibiotics: ampicillin, penicillin and amoxicillin. The cations were derived from substituted ammonium, phosphonium pyridinium and methylimidazolium salts, such as: tetraethyl ammonium, trihexiltetradecilphosphonium, cetylpyridinium, choline (an essential nutrient), 1-ethyl-3-methylimidazolium, and 1-ethanol-3-methyl imidazolium structures. Commercial ammonium and phosponium halogen salts were first transformed into hydroxides. on ionic exchange column (Amberlite IRA-400) in methanol. The prepared hydroxides were then neutralized with beta-lactam antibiotics. After crystallization we obtained pure ILs and salts containing beta-lactam antibiotics. This work presents a novel method for preparation of new salts of antibiotics with low melting point and their characterization.

Development and biological evaluation of API-ILs based on anti-bacterial and anti-fungal drugs

In recent years Ionic Liquids (ILs) are being applied in life sciences. ILs are being produce with active pharmaceutical drugs (API) as they can reduce polymorphism and drug solubility problems [1] Also ILs are being applied as a drug delivery device in innovative therapies What is appealing in ILs is the ILs building up platform, the counter-ion can be carefully chosen in order to avoid undesirable side effects or to give innovative therapies in which two active ions are paired. This work shows ILs based on ampicillin (an anti-bacterial agent) and ILs based on Amphotericin B. Also we show studies that indicate that ILs based on Ampicillin could reverse resistance in some bacteria. The ILs produced in this work were synthetized by the neutralization method described in Ferraz et. al. [2] Ampicillin anion was combined with the following organic cations 1-ethyl-3-methylimidazolium, [EMIM]; 1-hydroxy-ethyl-3-methylimidazolium, [C2OHMIM]; choline, [cholin]; tetraethylammonium, [TEA]; cetylpyridinium, [C16pyr] and trihexyltetradecylphosphonium, [P6,6,6,14]. Amphotericin B was combined with [C16pyr], [cholin] and 1-metohyethyl-3-methylimidazolium, [C3OMIM]. The ILs-APIs based on ampicillin[2] were tested against sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumonia (clinical isolated), as well as on Gram positive Staphylococcus Aureus ATCC 25923, Staphylococcus epidermidis and Enterococcus faecalis. The arising resistance developed by bacteria to antibiotics is a serious public health threat and needs new and urgent measures. We study the bacterial activity of these compounds against a panel of resistant bacteria (clinical isolated strains): E. coli CTX M9, E. coli TEM CTX M9, E. coli TEM1, E. coli CTX M2, E. coli AmpC Mox2. In this work we demonstrate that is possible to produce ILs from anti-bacterial and anti-fungal compounds. We show here that the new ILs can reverse the bacteria resistance. With the careful choice of the organic cation, it is possible to create important biological and physic-chemical properties. This work also shows that the ion-pair is fundamental in ampicillin mechanism of action.

ß-Lactams: chemical structure, mode of action and mechanisms of resistance

This synopsis summarizes the key chemical and bacteriological characteristics of β-lactams, penicillins, cephalosporins, carbanpenems, monobactams and others. Particular notice is given to first-generation to fifth-generation cephalosporins. This review also summarizes the main resistance mechanism to antibiotics, focusing particular attention to those conferring resistance to broad-spectrum cephalosporins by means of production of emerging cephalosporinases (extended-spectrum β-lactamases and AmpC β-lactamases), target alteration (penicillin-binding proteins from methicillin-resistant Staphylococcus aureus) and membrane transporters that pump β-lactams out of the bacterial cell.

ß-lactamases in the biochemistry and molecular biology laboratory

β-lactamases are hydrolytic enzymes that inactivate the β-lactam ring of antibiotics such as penicillins and cephalosporins. The major diversity of studies carried out until now have mainly focused on the characterization of β-lactamases recovered among clinical isolates of Gram-positive staphylococci and Gram-negative enterobacteria, amongst others. However, only some studies refer to the detection and development of β-lactamases carriers in healthy humans, sick animals, or even in strains isolated from environmental stocks such as food, water, or soils. Considering this, we proposed a 10-week laboratory programme for the Biochemistry and Molecular Biology laboratory for majors in the health, environmental, and agronomical sciences. During those weeks, students would be dealing with some basic techniques such as DNA extraction, bacterial transformation, polymerase chain reaction (PCR), gel electrophoresis, and the use of several bioinformatics tools. These laboratory exercises would be conducted as a mini research project in which all the classes would be connected with the previous ones. This curriculum was compared in an experiment involving two groups of students from two different majors. The new curriculum, with classes linked together as a mini research project, was taught to a major in Pharmacy and an old curriculum was taught to students from environmental health. The results showed that students who were enrolled in the new curriculum obtained better results in the final exam than the students who were enrolled in the former curriculum. Likewise, these students were found to be more enthusiastic during the laboratory classes than those from the former curriculum.

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

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