Complexation of beta-lactam antibiotic drug carbenicillin to bovine serum albumin: energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 831-840, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Development of biomass proportions in Norway spruce (Picea abies [L.] Karst.)

This study tests the hypotheses that (1) the above-ground structure of Norway spruce (Picea abies [L] Karst.) is derivable from the functional balance theory, and that (2) crown ratio is a key source of structural variation in trees of different age and social position. Twenty-nine trees were measured in three stands (young, middle-aged, and mature), with three thinning treatments (unthinned, normal, and intensive) in the two older stands. There was a strong linear relationship between the total cross-sectional area of branches and that of stem at crown base. Foliage mass was linearly related with stem basal area at crown base. Also an allometric relationship was found between foliage mass and crown length. The mean length (weighted by basal area) of branches obeyed an exponential function of crown length. The parameters of most of these relationships were independent of slenderness (tree height/breast height diameter) and tree age However, total branch cross-sectional area per stem cross-sectional area in the young trees was greater than in the older trees. The young trees also had slightly shorter branches than predicted by the mean branch length equation. This was probably caused by branch senescence which had not yet started in the young stand. The older trees had a relatively long lower crown segment which was growing slowly and senescing. It was proposed that a segmented crown structure is characteristic of shade tolerant tree species, and that the structural model could be further developed by making the two segments explicit.     

Counterintuitive size patterns in bivoltine moths: late-season larvae grow larger despite lower food quality

Within a season, successive generations of short-lived organisms experience different combinations of environmental parameters, such as temperature, food quality and mortality risk. Adult body size of e.g. insects is therefore expected to vary both as a consequence of proximate environmental effects as well as adaptive responses to seasonal cues. In this study, we examined intraspecific differences in body size between successive generations in 12 temperate bivoltine moths (Lepidoptera), with the ultimate goal to critically compare the role of proximate and adaptive mechanisms in determining seasonal size differences. In nearly all species, individuals developing late in the season (diapausing generation) attained a larger adult size than their conspecifics with the larval period early in the season (directly developing generation) despite the typically lower food quality in late summer. Rearing experiments conducted on one of the studied species, Selenia tetralunaria also largely exclude the possibility that the proximate effects of food quality and temperature are decisive in determining size differences between successive generations. Adaptive explanations appear likely instead: the larger body size in the diapausing generation may be adaptively associated with the lower bird predation pressure late in the season, and/or the likely advantage of large pupal size during overwintering.     

Utilization of intrachain 4'-C-azidomethylthymidine for preparation of oligodeoxyribonucleotide conjugates by click chemistry in solution and on a solid support

4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-[N-(trifluoroacetyl)aminomethyl]benzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.     

Molecular epidemiology of drug-resistant tuberculosis in Sweden

Drug-resistant tuberculosis (TB), including the more severe forms of multidrug- and extensively drug-resistant forms, is an increasing public health concern globally. In Sweden the majority of patients with TB are immigrants from countries with a high incidence of TB including the drug-resistant forms. In this study, the spread of resistant TB in Sweden was investigated by molecular fingerprinting. Isolates resistant to at least one of the drugs, isoniazid, rifampicin, ethambutol or streptomycin, from 400 patients collected between 1994 and 2005, were studied by restriction fragment length polymorphism (RFLP) and by spoligotyping. Thirty-five clusters of patients infected with strains with identical RFLP and spoligotyping patterns (2-96 patients per cluster), comprising a total of 203 patients, were found. One large outbreak of isoniazid resistant tuberculosis was identified, involving 96 patients, mainly from the Horn of Africa. To identify chains of transmission, molecular epidemiological characterization of TB isolates should, if possible, be performed on isolates from all new TB patients.     

11C]cyclopropyl-FLB 457: a PET radioligand for low densities of dopamine D2 receptors

(S)-5-bromo-N-[(1-cyclopropylmethyl-2-pyrrolidinyl)methyl]-2,3-dimethoxybenzamide (4) has pico-molar in vitro binding affinity to D(2) receptor (K(i) (D(2))=0.003 nM) with lower affinity to D(3) receptor (K(i) (D(3))=0.22 nM). In this study, we describe radiosynthesis of [(11)C]4 and evaluation of its binding characteristics in post-mortem human brain autoradiography and with PET in cynomolgus monkeys. The (11)C labelled 4 was synthesized by using [(11)C]methyltriflate in a methylation reaction with its phenolic precursor with good incorporation yield (64+/-11%, DCY) and high specific radioactivity >370 GBq/micromol (>10,000 Ci/mmol). In post-mortem human brain autoradiography [(11)C]4 exhibited high specific binding in brain regions enriched with dopamine D(2)/D(3) receptors and low level of non-specific binding. In cynomolgus monkeys [(11)C]4 exhibited high brain uptake reaching 4.4% ID at 7.5 min. The binding in the extrastriatal low density D(2)-receptor regions; thalamus and frontal, parietal, temporal, and occipital cortex, was clearly visible. Pre-treatment with raclopride (1 mg/kg as tartrate) caused high reduction of binding in extrastriatal regions, including cerebellum. [(11)C]4 is a promising radioligand for imaging D(2) receptors in low density regions in brain.     

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.