Chemical treatment of Escherichia coli: 3. Selective extraction of a recombinant protein from cytoplasmic inclusion bodies in intact cells

In previous parts of this study we developed procedures for the high-efficiency chemical extraction of soluble and insoluble protein from intact Escherichia coli cells. Although high yields were obtained, extraction of recombinant protein directly from cytoplasmic inclusion bodies led to low product purity due to coextraction of soluble contaminants. In this work, a two-stage procedure for the selective extraction of recombinant protein at high efficiency and high purity is reported. In the first stage, inclusion-body stability is promoted by the addition of 15 mM 2-hydroxyethyldisulfide (2-HEDS), also known as oxidized P-mercaptoethanol, to the permeabil ization buffer (6 M urea + 3 mM ethylenediaminetetra-acetate [EDTA]). 2-HEDS is an oxidizing agent believed to promote disulfide bond formation, rendering the inclusion body resistant to solubilization in 6 M urea. Contaminating proteins are separated from the inclusion-body fraction by centrifugation. in the second stage, disulfide bonds are readily eliminated by including reducing agent (20 mM dithiothreitol [DTT]) into the permeabilization buffer. Extraction using this selective two-stage process yielded an 81% (w/w) recovery of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact E. coli, at a purity of 46% (w/w). This was comparable to that achieved by conventional extraction (mechanical disruption followed by centrifugation and solubilization). A pilot-scale procedure was also demonstrated using a stirred reactor and diafiltration. This is the first reported study that achieves both high extraction efficiency and selectivity by the chemical treatment of cytoplasmic inclusion bodies in intact bacterial cells. (C) 1999 John Wiley & Sons, Inc.

Frequency stabilised grating feedback laser diode for atom cooling applications

The free running linewidth of an external cavity grating feedback diode laser is on the order of a few megahertz and is limited by the mechanical and acoustic vibrations of the external cavity. Such frequency fluctuations can be removed by electronic feedback. We present a hybrid stabilisation technique that uses both a Fabry-Perot confocal cavity and an atomic resonance to achieve excellent short and long term frequency stability. The system has been shown to reduce the laser linewidth of an external cavity diode laser by an order of magnitude to 140 kHz, while limiting frequency excursions to 60 kHz relative to an absolute reference over periods of several hours. The scheme also presents a simple way to frequency offset two lasers many gigahertz apart which should find a use in atom cooling experiments, where hyperfine ground-state frequency separations are often required.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Spl. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity. (C) 2000 Elsevier Science Ltd, All rights reserved.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein

Background-In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. Methods and Results-Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 2-1/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC50 [mol/L] NE 5.5+/-0.1, n=12; -EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC50 [mol/L] NE 5.8+/--0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC50 [mol/L] NE 5.7+/-0.2: EPI 5.8+/-0.1), Ventricular membranes from Fallot infants, labeled with (-)-[I-125]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. Conclusions-Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

The potential for a selective screening strategy for abdominal aortic aneurysm

Objectives-To investigate the feasibility of selective screening for abdominal aortic aneurysm (AAA) based on identification of a target group of manageable size defined by risk factors for AAA. Setting-Male residents of Perth, Western Australia, aged 65-83 years, who participated in a randomised controlled trial of ultrasound screening for AAA. Methods-Eligible men were identified from the electoral roll and invited to attend a screening clinic. Those who attended completed a questionnaire, had a limited physical examination, and underwent an ultrasound examination to identify the maximum diameter of the infrarenal aorta. Data on risk factors collected from the first 8995 men seen were used to calculate a multivariate risk score for the remaining 2755 men who were screened. Gentiles of the risk score were used to define potential target groups for screening and the sensitivity and specificity of each of these selective screening strategies were calculated. We repeated the calculation separately for AAAs of at least 30 mm, 40 mm, and 50 mm in diameter. Results-We found that screening half of the male population aged 65-83 years would find approximately 75% of AAAs, regardless of their size, whereas screening only current smokers in this population would find approximately 20% of AAAs. Conclusions-Selective screening for AAA using easily recognisable risk factors is feasible but is not worthwhile as approximately 25% of clinically significant cases would be missed.

Bond strength of an adhesive system irradiated with Nd : YAG laser in dentin treated with Er : YAG laser

The purpose of this in vitro study was to verify through micro tensile bond test the bond strength of an adhesive system irradiated with Nd:YAG laser in dentine previously treated with Er:YAG laser. Twenty caries free extracted human third molars were used. The teeth were divided in four experimental groups (n = 5): (G1) control group; (G2) irradiation of the adhesive system with the Nd:YAG laser; (G3) dentin treatment with Er:YAG laser; (G4) dentin treatment with Er:YAG laser followed by the irradiation of the adhesive system with Nd:YAG laser. The Er:YAG laser fluency parameter for the dentin treatment was of 60 J/cm(2). ne adhesive system was irradiated with the Nd:YAG laser with fluency of 100 J/cm(2). Dental restorations were performed with Adper Single Bond 2/Z250. One tooth from each group was prepared for the evaluation of the adhesive interface under SEM and bond failure tests were also performed and evaluated. The statistical analysis showed statistical significant difference between the groups G1 and G3, G1 and G4, G2 and G3, and G2 and G4; and similarity between the groups G1 and G2, and G3 and G4. The adhesive failures were predominant in all the experimental groups. The SEM analysis showed an adhesive interface with features confirming the results of the mechanical tests. The Nd:YAG laser on the adhesive system did not influence the bond strength in dentin treated or not with the Er:YAG laser.

Cost-effectiveness of cognitive behavioural therapy and selective serotonin reuptake inhibitors for major depression in children and adolescents

Objective: To assess from a health sector perspective the incremental cost-effectiveness of cognitive behavioural therapy (CBT) and selective serotonin reuptake inhibitors (SSRIs) for the treatment of major depressive disorder (MDD) in children and adolescents, compared to 'current practice'. Method: The health benefit is measured as a reduction in disability-adjusted life years (DALYs), based on effect size calculations from meta-analysis of randomised controlled trials. An assessment on second stage filter criteria ('equity'; 'strength of evidence', 'feasibility' and 'acceptability to stakeholders') is also undertaken to incorporate additional factors that impact on resource allocation decisions. Costs and benefits are tracked for the duration of a new episode of MDD arising in eligible children (age 6-17 years) in the Australian population in the year 2000. Simulation-modelling techniques are used to present a 95% uncertainty interval (UI) around the cost-effectiveness ratios. Results: Compared to current practice, CBT by public psychologists is the most cost-effective intervention for MDD in children and adolescents at A$9000 per DALY saved (95% UI A$3900 to A$24 000). SSRIs and CBT by other providers are less cost-effective but likely to be less than A$50 000 per DALY saved (> 80% chance). CBT is more effective than SSRIs in children and adolescents, resulting in a greater total health benefit (DALYs saved) than could be achieved with SSRIs. Issues that require attention for the CBT intervention include equity concerns, ensuring an adequate workforce, funding arrangements and acceptability to various stakeholders. Conclusions: Cognitive behavioural therapy provided by a public psychologist is the most effective and cost-effective option for the first-line treatment of MDD in children and adolescents. However, this option is not currently accessible by all patients and will require change in policy to allow more widespread uptake. It will also require 'start-up' costs and attention to ensuring an adequate workforce.

Subtype-selective noncompetitive or competitive inhibition of human alpha(1)-adrenergic receptors by rho-TIA

The 19-amino acid conopeptide (rho-TIA) was shown previously to antagonize noncompetitively alpha(1B)-adrenergic receptors (ARs). Because this is the first peptide ligand for these receptors, we compared its interactions with the three recombinant human alpha(1)-AR subtypes (alpha(1A), alpha(1B), and alpha(1D)). Radioligand binding assays showed that rho-TIA was 10-fold selective for human alpha(1B)- over alpha(1A)- and alpha(1D)-ARs. As observed with hamster alpha(1B)-ARs, rho-TIA decreased the number of binding sites (B-max) for human alpha(1B)-ARs without changing affinity (K-D), and this inhibition was unaffected by the length of incubation but was reversed by washing. However, rho-TIA had opposite effects at human alpha(1A)-ARs and alpha(1D)-ARs, decreasing KD without changing Bmax, suggesting it acts competitively at these subtypes. rho-TIA reduced maximal NE-stimulated [H-3] inositol phosphate formation in HEK293 cells expressing human alpha(1B)-ARs but competitively inhibited responses in cells expressing alpha(1A)- or alpha(1D)-ARs. Truncation mutants showed that the amino-terminal domains of alpha(1B)- or alpha(1D)-ARs are not involved in interaction with rho-TIA. Alanine-scanning mutagenesis of rho-TIA showed F18A had an increased selectivity for alpha(1B)-ARs, and F18N also increased subtype selectivity. I8A had a slightly reduced potency at alpha(1B)-ARs and was found to be a competitive, rather than noncompetitive, inhibitor in both radioligand and functional assays. Thus rho-TIA noncompetitively inhibits alpha(1B)-ARs but competitively inhibits the other two subtypes, and this selectivity can be increased by mutation. These differential interactions do not involve the receptor amino termini and are not because of the charged nature of the peptide, and isoleucine 8 is critical for its noncompetitive inhibition at alpha(1B)-ARs.

Er(3+)-doped silica-hafnia films for optical waveguides and spherical resonators

In this paper we present some result on sol-gel derived silica-hafnia systems. In particular we focus on fabrication, morphological and spectroscopic assessment of Er(3+)-activated thin films. Two examples of silica-hafnia-derived waveguiding glass ceramics, prepared by top-down and bottom-up techniques are reported, and the main optical properties are discussed. Finally, some properties of activated microspherical resonators, having a silica core, obtained by melting the end of a telecom fiber, coated with an Er(3+)-doped 70SiO(2)-30HfO(2) film, are presented. (C) 2009 Elsevier B.V. All rights reserved.

Selective involvement of GABAergic mechanisms of the dorsal periaqueductal gray and inferior colliculus on the memory of the contextual fear as assessed by the fear potentiated startle test

The inferior colliculus (IC) together with the dorsal periaqueductal gray (dPAG), the amygdala and the medial hypothalamus make part of the brain aversion system, which has mainly been related to the organization of unconditioned fear. However, the involvement of the IC and dPAG in the conditioned fear is still unclear. It is certain that GABA has a regulatory role on the aversive states generated and elaborated in these midbrain structures. In this study, we evaluated the effects of injections of the GABA-A receptor agonist muscimol (1.0 and 2.0 nmol/0.2 mu L) into the IC or dPAG on the freezing and fear-potentiated startle (FPS) responses of rats submitted to a context fear conditioning. Intra-IC injections of muscimol did not cause any significant effect on the FPS or conditioned freezing but enhanced the startle reflex in non-conditioned animals. In contrast, intra-dPAG injections of muscimol caused significant reduction in FPS and conditioned freezing without changing the startle reflex in non-conditioned animals. Thus, intra-dPAG injections of muscimol produced the expected inhibitory effects on the anxiety-related responses, the FPS and the freezing whereas these injections into the IC produced quite opposite effects suggesting that descending inhibitory pathways from the IC, probably mediated by GABA-A mechanisms, exert a regulatory role on the lower brainstem circuits responsible for the startle reflex. (C) 2008 Elsevier Inc. All rights reserved.

Midazolam reduces the selective activation of the rhinal cortex by contextual fear stimuli

Independent brain circuits appear to underlie different forms of conditioned fear, depending on the type of conditioning used, such as a context or explicit cue paired with footshocks. Several clinical reports have associated damage to the medial temporal lobe (MTL) with retrograde amnesia. Although a number of studies have elucidated the neural circuits underlying conditioned fear, the involvement of MTL components in the aversive conditioning paradigm is still unclear. To address this issue, we assessed freezing responses and Fos protein expression in subregions of the rhinal cortex and ventral hippocampus of rats following exposure to a context, light or tone previously paired with footshock (Experiment 1). A comparable degree of freezing was observed in the three types of conditioned fear, but with distinct patterns of Fos distribution. The groups exposed to cued fear conditioning did not show changes in Fos expression, whereas the group subjected to contextual fear conditioning showed selective activation of the ectorhinal (Ect), perirhinal (Per), and entorhinal (Ent) cortices, with no changes in the ventral hippocampus. We then examined the effects of the benzodiazepine midazolam injected bilaterally into these three rhinal subregions in the expression of contextual fear conditioning (Experiment 2). Midazolam administration into the Ect, Per, and Ent reduced freezing responses. These findings suggest that contextual and explicit stimuli endowed with aversive properties through conditioning recruit distinct brain areas, and the rhinal cortex appears to be critical for storing context-, but not explicit cue-footshock, associations. (C) 2010 Elsevier B.V. All rights reserved.

Evaluation of the effects of Candidatus Liberibacter asiaticus on inoculated citrus plants using laser-induced breakdown spectroscopy (LIBS) and chemometrics tools

This study investigated the organic and inorganic constituents of healthy leaves and Candidatus Liberibacter asiaticus (CLas)-inoculated leaves of citrus plants. The bacteria CLas are one of the causal agents of citrus greening (or Huanglongbing) and its effect on citrus leaves was investigated using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics. The information obtained from the LIBS spectra profiles with chemometrics analysis was promising for the construction of predictive models to identify healthy and infected plants. The major, macro- and microconstituents were relevant for differentiation of the sample conditions. The models were then applied to different inoculation times (from 1 to 8 months). The models were effective in the classification of 82-97% of the diseased samples with a 95% significance level. The novelty of this method was in the fingerprinting of healthy and diseased plants based on their organic and inorganic contents. (C) 2010 Elsevier B.V. All rights reserved.

Effect of Diode-Laser and AC Magnetic Field of Bovine Serum Albumin Nanospheres Loaded with Phthalocyanine and Magnetic Particles

This study reports on the development and characterization of bovine serum albumin (BSA) nanospheres containing Silicon(IV) phthalocyanine (NzPc) and/or maghemite nanoparticles (MNP), the latter introduced via ionic magnetic fluid (MF). The nanosized BSA-loaded samples were designed for synergic application while combining Photodynamic Therapy and Hyperthermia. Incorporation of MNP in the albumin-based template, allowing full control of the magnetic content, was accomplished by adding a highly-stable ionic magnetic fluid sample to the albumin suspension, following heat denaturing. The material`s evaluation was performed using Zeta potential measurements and scanning electron microscopy. The samples were characterized by steady-state techniques and time-resolved fluorescence. The in vitro assay, using human fibroblasts, revealed no cytotoxic effect in all samples investigated, demonstrating the potential of the tested system as a synergistic drug delivery system.