Control of targeting ligand display by pH-responsive polymers on gold nanoparticles mediates selective entry into cancer cells.

Selective targeting of cells for intracellular delivery of therapeutics represents a major challenge for pharmaceutical intervention in disease. Here we show pH-triggered receptor-mediated endocytosis of nanoparticles via surface ligand exposure. Gold nanoparticles were decorated with two polymers: a 2 kDa PEG with a terminal folate targeting ligand, and a di-block copolymer including a pH-responsive and a hydrophilic block. At the normal serum pH of 7.4, the pH-responsive block (apparent pKa of 7.1) displayed a hydrophilic extended conformation, shielding the PEG-folate ligands, which inhibited cellular uptake of the nanoparticles. Under pH conditions resembling those of the extracellular matrix around solid tumours (pH 6.5), protonation of the pH-responsive polymer triggered a coil-to-globule polymer chain contraction, exposing folate residues on the PEG chains. In line with this, endocytosis of folate-decorated polymer-coated gold nanoparticles in cancer cells overexpressing folate receptor was significantly increased at pH 6.5, compared with pH 7.4. Thus, the tumour acidic environment and high folate receptor expression were effectively exploited to activate cell binding and endocytosis of these nanoparticles. These data provide proof-of-concept for strategies enabling extracellular pH stimuli to selectively enhance cellular uptake of drug delivery vectors and their associated therapeutic cargo.

Premenopausal abnormal uterine bleeding and risk of endometrial cancer.

BACKGROUND: Endometrial biopsies are undertaken in premenopausal women with abnormal uterine bleeding but the risk of endometrial cancer or atypical hyperplasia is unclear. OBJECTIVES: To conduct a systematic literature review to establish the risk of endometrial cancer and atypical hyperplasia in premenopausal women with abnormal uterine bleeding. SEARCH STRATEGY: Search of PubMed, Embase and the Cochrane Library from database inception to August 2015. SELECTION CRITERIA: Studies reporting rates of endometrial cancer and/or atypical hyperplasia in women with premenopausal abnormal uterine bleeding. DATA COLLECTION AND ANALYSIS: Data were independently extracted by two reviewers and cross-checked. For each outcome, the risk and a 95% CI were estimated using logistic regression with robust standard errors to account for clustering by study. MAIN RESULTS: Sixty-five articles contributed to the analysis. Risk of endometrial cancer was 0.33% (95% CI 0.23-0.48%, n = 29 059; 97 cases) and risk of endometrial cancer or atypical hyperplasia was 1.31% (95% CI 0.96-1.80, n = 15 772; 207 cases). Risk of endometrial cancer was lower in women with heavy menstrual bleeding (HMB) (0.11%, 95% CI 0.04-0.32%, n = 8352; 9 cases) compared with inter-menstrual bleeding (IMB) (0.52%, 95% CI 0.23-1.16%, n = 3109; 14 cases). Of five studies reporting the rate of atypical hyperplasia in women with HMB, none identified any cases. CONCLUSIONS: The risk of endometrial cancer or atypical hyperplasia in premenopausal women with abnormal uterine bleeding is low. Premenopausal women with abnormal uterine bleeding should first undergo conventional medical management. Where this fails, the presence of IMB and older age may be indicators for further investigation. Further research into the risks associated with age and the cumulative risk of co-morbidities is needed. TWEETABLE ABSTRACT: Contrary to practice, premenopausal women with heavy periods or inter-menstrual bleeding rarely require biopsy.

Health locus of control beliefs and smoking among male Kuwaiti government employees.

A cross-sectional multistage stratified cluster survey of 1798 Kuwaiti male adults investigated the relationship between health locus of control (HLC) beliefs, health beliefs about smoking and smoking status. Non-smokers had stronger external HLC and better health beliefs about smoking than smokers. Thus the Kuwaiti men who used tobacco believed themselves to be more in control of their own lives than did non-users. Stepwise logistic regression analysis revealed that low HLC, poor health beliefs about smoking, single marital status and low level of education were significant predictors of risk of smoking. Simple linear regression analysis showed a significant negative relationship between HLC and health beliefs about smoking among non-smokers and ex-smokers, but not among smokers.

Health locus of control beliefs and smoking among male Kuwaiti government employees

A cross-sectional multistage stratified cluster survey of 1798 Kuwaiti male adults investigated the relationship between health locus of control [HLC] beliefs, health beliefs about smoking and smoking status. Non-smokers had stronger external HLC and better health beliefs about smoking than smokers. Thus the Kuwaiti men who used tobacco believed themselves to be more in control of their own lives than did non-users. Stepwise logistic regression analysis revealed that low HLC, poor health beliefs about smoking, single marital status and low level of education were significant predictors of risk of smoking. Simple linear regression analysis showed a significant negative relationship between HLC and health beliefs about smoking among non-smokers and ex-smokers, but not among smokers

Effects of magnesium sulphate on cerebral haemodynamics in healthy volunteers: a transcranial Doppler study.

BACKGROUND: Magnesium is increasingly being considered as a neuroprotective agent. We aimed to study its effects on middle cerebral artery blood flow velocity (V(mca)), cerebral autoregulation and cerebral vascular reactivity to carbon dioxide (CRCO(2)) in healthy volunteers. METHODS: Fifteen healthy volunteers were recruited. Using transcranial Doppler ultrasonography, V(mca) was recorded continuously. The strength of autoregulation was assessed by the transient hyperaemic response test, and the CRCO(2) was measured by assessing changes in V(mca) to the induced changes in end-tidal carbon dioxide. I.V. infusion of magnesium sulphate was then started (loading dose of 16 mmol followed by an infusion at the rate of 2.7 mmol h(-1)) for 45 min. The cerebral haemodynamic variables were measured again near the end of the infusion of magnesium sulphate. RESULTS: Total serum magnesium levels were doubled by the infusion regimen. However, there were no significant changes in V(mca), strength of autoregulation, or CRCO(2). Five of the volunteers reported marked nausea and two developed significant hypotension during the loading dose. CONCLUSIONS: Infusion of magnesium sulphate, in a dose that doubles its concentration in plasma, does not affect V(mca), strength of autoregulation or CRCO(2) in healthy volunteers. However, it can be associated with nausea and hypotension.

Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin.

The side chain of residue Arg(238) in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg(238) in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg(238)-->Lys), the positive charge was removed (Arg(238)-->Met) or the charge was reversed (Arg(238)-->Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E(12)) for each enzyme fall within the region of -247+/-10 mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme-NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy-enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.

Degradation of explosives by nitrate ester reductases.

Explosive-contaminated land poses a hazard both to the environment and to human health. Microbial enzymes, either in their native or heterologous hosts, are a powerful and low-cost tool for eliminating this environmental hazard. As many explosives have only been present in the environment for 10 years, and with similar molecules not known in Nature, the origin of enzymes specialized for the breakdown of explosives is of particular interest. Screening of environmental isolates resulted in the discovery of flavoproteins capable of denitrating the explosives pentaerythritol tetranitrate (PETN) and glycerol trinitrate. These nitrate ester reductases are related in sequence and structure to Old Yellow Enzyme from Saccharomyces carlsbergenisis. All the members of this family have alpha/beta barrel structures and FMN as a prosthetic group, and reduce various electrophilic substrates. The nitrate ester reductases are, however, unusual in that they display activity towards the highly recalcitrant, aromatic explosive 2,4,6-trinitrotoluene, via a reductive pathway resulting in nitrogen liberation. We have embarked on a detailed study of the structure and mechanism of PETN reductase from a strain of Enterobacter cloacae. Work is focused currently on relating structure and function within this growing family of enzymes, with a view to engineering novel enzymes exhibiting useful characteristics.

Relationship of aneurysmal subarachnoid hemorrhage to changes in atmospheric pressure: results of a prospective study.

OBJECT: The purpose of this study was to evaluate the relationship between atmospheric pressure and subarachnoid hemorrhage (SAH) in a region in the English Midlands. METHODS: All patients with angiographically proven SAH for the calendar year 1998 were analyzed. A geographical allocation was made based on the patients' origin within the region. The events were then compared with the data available for the local atmospheric pressures. One hundred nine patients had an SAH during the time period studied. The median atmospheric pressure recorded was 1014.5 millibars. Atmospheric pressure was modestly correlated with the number of SAHs per day (Spearman's rank correlation, r = 0.33; p < 0.0001); the daily change in atmospheric pressure also correlated mildly (r = 0.34, p < 0.0001). No other statistically significant association was found. CONCLUSIONS: The authors have shown a relationship between high atmospheric pressure and increased incidence of SAH. The underlying reason for this remains obscure.

Crystal structure of pentaerythritol tetranitrate reductase: "flipped" binding geometries for steroid substrates in different redox states of the enzyme.

Pentaerythritol tetranitrate reductase (PETN reductase) degrades high explosive molecules including nitrate esters, nitroaromatics and cyclic triazine compounds. The enzyme also binds a variety of cyclic enones, including steroids; some steroids act as substrates whilst others are inhibitors. Understanding the basis of reactivity with cyclic enones requires structural information for the enzyme and key complexes formed with steroid substrates and inhibitors. The crystal structure of oxidised and reduced PETN reductase at 1.5 A resolution establishes a close structural similarity to the beta/alpha-barrel flavoenzyme, old yellow enzyme. In complexes of oxidised PETN reductase with progesterone (an inhibitor), 1,4-androstadiene-3,17-dione and prednisone (both substrates) the steroids are stacked over the si-face of the flavin in an orientation different from that reported for old yellow enzyme. The specifically reducible 1,2 unsaturated bonds in 1,4-androstadiene-3,17-dione and prednisone are not optimally aligned with the flavin N5 in oxidised enzyme complexes. These structures suggest either relative "flipping" or shifting of the steroid with respect to the flavin when bound in different redox forms of the enzyme. Deuterium transfer from nicotinamide coenzyme to 1,4-androstadiene-3,17-dione via the enzyme bound FMN indicates 1alpha addition at the steroid C2 atom. These studies rule out lateral motion of the steroid and indicate that the steroid orientation is "flipped" in different redox states of the enzyme.

DNA-binding mechanism of the Escherichia coli Ada O(6)-alkylguanine-DNA alkyltransferase.

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O:(6)-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151-160) which form the recognition helix and the 'wing' of a helix-turn-wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O:(6)-methylguanine (O:(6)meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain.

Serine acetyltransferase from Escherichia coli is a dimer of trimers.

Equilibrium sedimentation studies show that the serine acetyltransferase (SAT) of Escherichia coli is a hexamer. The results of velocity sedimentation and quasi-elastic light scattering experiments suggest that the identical subunits are loosely packed and/or arranged in an ellipsoidal fashion. Chemical cross-linking studies indicate that the fundamental unit of quaternary structure is a trimer. The likelihood, therefore, is that in solution SAT exists as an open arrangement of paired trimers. Crystals of SAT have 32 symmetry, consistent with such an arrangement, and the cell density function is that expected for a hexamer. Electron microscopy with negative staining provides further evidence that SAT has an ellipsoidal subunit organization, the dimensions of the particles consistent with the proposed paired trimeric subunit arrangement. A bead model analysis supports the view that SAT has a low packing density and, furthermore, indicates that the monomers may have an ellipsoidal shape. Such a view is in keeping with the ellipsoidal subunit shape of trimeric LpxA, an acyltransferase with which SAT shares contiguous repeats of a hexapeptide motif.

Epidemiology of smoking among Kuwaiti adults: prevalence, characteristics, and attitudes.

INTRODUCTION: In 1996 we conducted a cross-sectional survey to study the epidemiology of smoking among Kuwaiti adults. METHODS: The 4000 participants were selected using a three-stage stratified cluster sampling design. Altogether 3859 participants (1798 males, 2061 females) returned a completed self-administered questionnaire. RESULTS: The prevalence of smoking was 34.4% (95% confidence interval (CI) = 32.2-36.6) among men and 1.9% (95% CI = 1.3-2.5) among women. Among men, the highest prevalence (56.5%; 95% CI = 36.2-76.8) was observed in the youngest age group (< or = 20 years). Among women the highest prevalence was observed in one of the older age groups (46-50 years) (7.1%; 95% CI = 3.1-11.1). Multiple logistic regression analysis showed that the following factors were independently associated with smoking: lower levels of education (odds ratio (OR) 3.5; 95% CI = 1.5-8.4), lower employment grade (OR = 4.1; 2.5-6.7), and being a separated, divorced, or widowed woman (OR = 4.9; 95% CI = 2.0-11.8). The majority of smokers (68%) began smoking when younger than 20 years; significantly more men (70%) than women (33%) began smoking at these ages (P < 0.0001). On average, men began smoking at an earlier age (18 years vs 21 years; P < 0.001) and therefore had smoked for a longer period (15 years vs 12 years; P < 0.05); men also consumed a higher number of cigarettes each day (26 vs 17; P < 0.05). A large proportion of smokers were ignorant about the health consequences of passive smoking: about 77% of those with children reported that they smoked in the presence of their children. Almost half (47%) of all smokers stated that they wanted to stop smoking, and about 56% had attempted to quit. The biggest perceived barrier to quitting was uncertainty about "how to quit". A total of 338 respondents (8.8%; 95% CI = 5.8-11.9) were classified as former smokers. About half of the former smokers had quit between the ages of 20 and 29 years; the average age of quitting was 28 years. Former smokers were more likely to have smoked fewer cigarettes per day and to have smoked for significantly less time than current smokers. DISCUSSION: Given the fact that free education is provided at all levels by the government, anti-tobacco education and awareness should be included as an integral part of the curriculum in schools and colleges.

BP: developing excellence in the global enterprise.

World-class supply management organizations like Honda of America, John Deere, Chrysler, Hewlett Packard, and Motorola all understand the power of continuous improvement in the supply base. The best supplier management and development programs help suppliers--on a long-term basis--develop their own strength and expertise in suggestion systems, new product development, and high quality production results.

Crystal structure of the human O(6)-alkylguanine-DNA alkyltransferase.

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to O(6)-alkylation of guanine in DNA. This lesion results in transition mutations. In both prokaryotic and eukaryotic cells, repair is effected by direct reversal of the damage by a suicide protein, O(6)-alkylguanine-DNA alkyltransferase. The alkyltransferase removes the alkyl group to one of its own cysteine residues. However, this mechanism for preserving genomic integrity limits the effectiveness of certain alkylating anticancer agents. A high level of the alkyltransferase in many tumour cells renders them resistant to such drugs. Here we report the X-ray structure of the human alkyltransferase solved using the technique of multiple wavelength anomalous dispersion. This structure explains the markedly different specificities towards various O(6)-alkyl lesions and inhibitors when compared with the Escherichia coli protein (for which the structure has already been determined). It is also used to interpret the behaviour of certain mutant alkyltransferases to enhance biochemical understanding of the protein. Further examination of the various models proposed for DNA binding is also permitted. This structure may be useful for the design and refinement of drugs as chemoenhancers of alkylating agent chemotherapy.

The structure of the exo-beta-(1,3)-glucanase from Candida albicans in native and bound forms: relationship between a pocket and groove in family 5 glycosyl hydrolases.

A group of fungal exo-beta-(1,3)-glucanases, including that from the human pathogen Candida albicans (Exg), belong to glycosyl hydrolase family 5 that also includes many bacterial cellulases (endo-beta-1, 4-glucanases). Family members, despite wide sequence variations, share a common mechanism and are characterised by possessing eight invariant residues making up the active site. These include two glutamate residues acting as nucleophile and acid/base, respectively. Exg is an abundant secreted enzyme possessing both hydrolase and transferase activity consistent with a role in cell wall glucan metabolism and possibly morphogenesis. The structures of Exg in both free and inhibited forms have been determined to 1.9 A resolution. A distorted (beta/alpha)8 barrel structure accommodates an active site which is located within a deep pocket, formed when extended loop regions close off a cellulase-like groove. Structural analysis of a covalently bound mechanism-based inhibitor (2-fluoroglucosylpyranoside) and of a transition-state analogue (castanospermine) has identified the binding interactions at the -1 glucose binding site. In particular the carboxylate of Glu27 serves a dominant hydrogen-bonding role. Access by a 1,3-glucan chain to the pocket in Exg can be understood in terms of a change in conformation of the terminal glucose residue from chair to twisted boat. The geometry of the pocket is not, however, well suited for cleavage of 1,4-glycosidic linkages. A second glucose site was identified at the entrance to the pocket, sandwiched between two antiparallel phenylalanine side-chains. This aromatic entrance-way must not only direct substrate into the pocket but also may act as a clamp for an acceptor molecule participating in the transfer reaction.

Analysis and characterization of data from twinned crystals.

It is difficult but not impossible to determine a macromolecular structure using X-ray data obtained from twinned crystals, providing it is noticed and corrected. For perfectly twinned crystals, the structure can probably only be solved by molecular replacement. It is possible to detect and characterize twinning from an analysis of the intensity statistics and crystal packing density. Tables of likely twinning operators and some examples are discussed here.

Crystal structure of Trematomus newnesi haemoglobin re-opens the root effect question.

As new structural data have become available, somewhat contrasting explanations of the Root effect in fish haemoglobins (Hb) have been provided. Hb 1 of the Antarctic fish Trematomus newnesi has a nearly pH-independent oxygen affinity, in spite of 95 % sequence identity with Hb 1 of Trematomus (previously named Pagothenia) bernacchii that has a strong Root effect. Here, the 2.2 A R-state structure of Trematomus newnesi Hb 1 is presented. The structure is similar to that of Root effect fish Hbs from Spot and T. bernacchii, suggesting that the differences in the pH dependence cannot be related to the modulation of the R-state. In comparison to T. bernacchii Hb 1, the role of the three mutations Thr41 (C6)alpha-->Ile, Ala97 (G3)alpha-->Ser and His41 (C7)beta-->Tyr at the alpha1beta2-interface is discussed.

Ligand-induced conformational change in penicillin acylase.

The enzyme penicillin acylase (penicillin amidohydrolase EC 3.5.1. 11) catalyses the cleavage of the amide bond in the benzylpenicillin (penicillin G) side-chain to produce phenylacetic acid and 6-aminopenicillanic acid (6-APA). The enzyme is of great pharmaceutical importance, as the product 6-APA is the starting point for the synthesis of many semi-synthetic penicillin antibiotics. Studies have shown that the enzyme is specific for hydrolysis of phenylacetamide derivatives, but is more tolerant of features in the rest of the substrate. It is this property that has led to many other applications for the enzyme, and greater knowledge of the enzyme's structure and specificity could facilitate engineering of the enzyme, enhancing its potential for chemical and industrial applications. An extensive study of the binding of a series of phenylacetic acid derivatives has been carried out. A measure of the relative degree of inhibition of the enzyme by each of the compounds has been obtained using a competitive inhibition assay, and the structures of a number of these complexes have been determined by X-ray crystallography. The structures reveal a clear rationale for the observed kinetic results, but show also that some of the ligands cause a conformational change within the binding pocket. This change can generally be understood in terms of the size and orientation of the ligand within the active site.The results reveal that ligand binding in penicillin acylase is facilitated by certain amino acid residues that can adopt two distinct, energetically favourable positions in order to accommodate a variety of compounds within the active site. The structures of these complexes provide evidence for conformational changes in the substrate-binding region that may act as a switch in the mechanism of autocatalytic processing of this enzyme.