Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.     

Cold acclimation increases basal heart rate but decreases its thermal tolerance in rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss, Walbaum) were acclimated to 4 degrees C and 17 degrees C for more than 4 weeks and heart rate was determined in the absence and presence of adrenaline to see how thermal adaptation influences basal heart rate and its beta-adrenergic control in a eurythermal fish species. The basal heart rate in vitro was higher in cold-acclimated than warm-acclimated rainbow trout at temperatures below 17 degrees C. On the other hand, adaptation to cold decreased thermal tolerance of heart rate so that the maximal heart rates were achieved at 17 degrees C (75 +/- 4 bpm) and 24 degrees C (88 +/- 2 bpm) in cold-acclimated and warm-acclimated trout, respectively. Beta-adrenergic response of the heart was enhanced by cold-adaptation, since adrenaline (100 nmol l(-1)) caused stronger stimulation of heart rate in cold-acclimated (29 +/- 14%) than in warm-acclimated fish (10 +/- 1%; P = 0.03). Furthermore, adrenaline strongly opposed the temperature-dependent deterioration of force production in cold-acclimated trout but not in warm-acclimated trout. The results indicate that adaptation to cold increases basal heart rate but decreases its thermal tolerance in rainbow trout. Cold acclimation up-regulates the beta-adrenergic system, and beta-adrenoceptor activation seems to provide cardioprotection against high temperatures in the cold-adapted rainbow trout.     

Xylitol purification by cross-linked glucose isomerase crystals

Xylitol is specifically bound by active, cross-linked glucose isomerase crystals (CLGI). CLGI can be used to purify xylitol or concentrate it from dilute and impure solutions. Bound xylitol can be eluted from CLGI by Ca2 and the material reactivated by Mg2. The binding capacity is 1 mg xylitol per 525 mg CLGI which equals one molecule per active center. CLGI can further be used to purify xylitol and sorbitol from impure mixtures of arabinitol, mannitol, ribitol and monosaccharides. © Rapid Science Ltd. 1998     

Effects of Auditory Attention Training with the Dichotic Listening Task: Behavioural and Neurophysiological Evidence

Facilitation of general cognitive capacities such as executive functions through training has stirred considerable research interest during the last decade. Recently we demonstrated that training of auditory attention with forced attention dichotic listening not only facilitated that performance but also generalized to an untrained attentional task. In the present study, 13 participants underwent a 4-week dichotic listening training programme with instructions to report syllables presented to the left ear (FL training group). Another group (n = 13) was trained using the non-forced instruction, asked to report whichever syllable they heard the best (NF training group). The study aimed to replicate our previous behavioural results, and to explore the neurophysiological correlates of training through event-related brain potentials (ERPs). We partially replicated our previous behavioural training effects, as the FL training group tended to show more allocation of auditory spatial attention to the left ear in a standard dichotic listening task. ERP measures showed diminished N1 and enhanced P2 responses to dichotic stimuli after training in both groups, interpreted as improvement in early perceptual processing of the stimuli. Additionally, enhanced anterior N2 amplitudes were found after training, with relatively larger changes in the FL training group in the forced-left condition, suggesting improved top-down control on the trained task. These results show that top-down cognitive training can modulate the left-right allocation of auditory spatial attention, accompanied by a change in an evoked brain potential related to cognitive control.     

IL-10 Gene Polymorphisms Are Associated with Post-Bronchiolitis Lung Function Abnormalities at Six Years of Age

Aim

Interleukin-10 (IL-10) has been associated with wheezing and asthma in children and the genetic variation of the IL-10 cytokine production may be linked to post-bronchiolitis lung function. We used impulse oscillometry (IOS) to evaluate the associations of IL10 polymorphisms with lung function at a median age of 6.3 years in children hospitalised for bronchiolitis before six months of age.

Methods

We performed baseline and post-exercise IOS on 103 former bronchiolitis patients. Data on single nucleotide polymorphisms (SNP) of IL10 rs1800896 (–1082G/A), rs1800871 (–819C/T), rs1800872 (–592C/A) were available for 99 children and of IL10 rs1800890 (–3575T/A) for 98 children.

Results

IL10 rs1800896, rs1800871 and rs1800872 combined genotype AA+CT+CA and carriage of haplotype ATA, respectively, were associated with higher resistance and lower reactance in baseline IOS in adjusted analyses. At IL10 rs1800890, the A/A-genotype and carriers of A-allele were associated with lower reactance in baseline IOS. There were no significant associations between the studied SNPs and airway hyper-reactivity to exercise.

Conclusion

Low-IL-10-producing polymorphisms in the IL-10 encoding gene were associated with obstructive lung function parameters, suggesting an important role for IL-10 in development of lung function deficit in early bronchiolitis patients.     

CMPF Does Not Associate with Impaired Glucose Metabolism in Individuals with Features of Metabolic Syndrome

Objective

3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF) is a metabolite produced endogenously from dietary sources of furan fatty acids. The richest source of furan fatty acids in human diet is fish. CMPF was recently shown to be elevated in fasting plasma in individuals with gestational diabetes and type 2 diabetes, and mechanistically high level of CMPF was linked to β cell dysfunction. Here we aimed to study the association between plasma CMPF level and glucose metabolism in persons with impaired glucose metabolism.

Methods

Plasma CMPF concentration was measured from plasma samples of the study participants in an earlier controlled dietary intervention. All of them had impaired glucose metabolism and two other characteristics of the metabolic syndrome. Altogether 106 men and women were randomized into three groups for 12 weeks with different fish consumption (either three fatty fish meals per week, habitual fish consumption or maximum of one fish meal per week). Associations between concentration of CMPF and various glucose metabolism parameters at an oral glucose tolerance test at baseline and at the end of the study were studied.

Results

Fasting plasma CMPF concentration was significantly increased after a 12-week consumption of fatty fish three times per week, but the concentration remained much lower compared to concentrations reported in diabetic patients. Increases of plasma CMPF concentrations mostly due to increased fish consumption were not associated with impaired glucose metabolism in this study. Instead, elevated plasma CMPF concentration was associated with decreased 2-hour insulin concentration in OGTT.

Conclusions

Moderately elevated concentration of CMPF in plasma resulting from increased intake of fish is not harmful to glucose metabolism. Further studies are needed to fully explore the role of CMPF in the pathogenesis of impaired glucose metabolism.

Trial Registration

ClinicalTrials.gov NCT00573781     

Structural analysis of the complex between calmodulin and full-length myelin basic protein, an intrinsically disordered molecule

Myelin basic protein (MBP) is present between the cytoplasmic leaflets of the compact myelin membrane in both the peripheral and central nervous systems, and characterized to be intrinsically disordered in solution. One of the best-characterized protein ligands for MBP is calmodulin (CaM), a highly acidic calcium sensor. We pulled down MBP from human brain white matter as the major calcium-dependent CaM-binding protein. We then used full-length brain MBP, and a peptide from rodent MBP, to structurally characterize the MBP–CaM complex in solution by small-angle X-ray scattering, NMR spectroscopy, synchrotron radiation circular dichroism spectroscopy, and size exclusion chromatography. We determined 3D structures for the full-length protein–protein complex at different stoichiometries and detect ligand-induced folding of MBP. We also obtained thermodynamic data for the two CaM-binding sites of MBP, indicating that CaM does not collapse upon binding to MBP, and show that CaM and MBP colocalize in myelin sheaths. In addition, we analyzed the post-translational modifications of rat brain MBP, identifying a novel MBP modification, glucosylation. Our results provide a detailed picture of the MBP–CaM interaction, including a 3D model of the complex between full-length proteins.     

Common genetic variants and modification of penetrance of BRCA2-associated breast cancer

The considerable uncertainty regarding cancer risks associated with inherited mutations of BRCA2 is due to unknown factors. To investigate whether common genetic variants modify penetrance for BRCA2 mutation carriers, we undertook a two-staged genome-wide association study in BRCA2 mutation carriers. In stage 1 using the Affymetrix 6.0 platform, 592,163 filtered SNPs genotyped were available on 899 young (<40 years) affected and 804 unaffected carriers of European ancestry. Associations were evaluated using a survival-based score test adjusted for familial correlations and stratified by country of the study and BRCA2*6174delT mutation status. The genomic inflation factor (λ) was 1.011. The stage 1 association analysis revealed multiple variants associated with breast cancer risk: 3 SNPs had p-values<10(-5) and 39 SNPs had p-values<10(-4). These variants included several previously associated with sporadic breast cancer risk and two novel loci on chromosome 20 (rs311499) and chromosome 10 (rs16917302). The chromosome 10 locus was in ZNF365, which contains another variant that has recently been associated with breast cancer in an independent study of unselected cases. In stage 2, the top 85 loci from stage 1 were genotyped in 1,264 cases and 1,222 controls. Hazard ratios (HR) and 95% confidence intervals (CI) for stage 1 and 2 were combined and estimated using a retrospective likelihood approach, stratified by country of residence and the most common mutation, BRCA2*6174delT. The combined per allele HR of the minor allele for the novel loci rs16917302 was 0.75 (95% CI 0.66-0.86, ) and for rs311499 was 0.72 (95% CI 0.61-0.85, ). FGFR2 rs2981575 had the strongest association with breast cancer risk (per allele HR = 1.28, 95% CI 1.18-1.39, ). These results indicate that SNPs that modify BRCA2 penetrance identified by an agnostic approach thus far are limited to variants that also modify risk of sporadic BRCA2 wild-type breast cancer.     

Highly Diverse Cyanobactins in Strains of the Genus Anabaena

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26).The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain d-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20).Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids.     

Molecular characterization of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> biovar <Emphasis Type="Italic">longum</Emphasis> NAL8 plasmids and construction of a novel replicon screening system

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users only.