Histochemical detection of the α-D-arabinopyranoside configuration using fluorescent-labelled concanavalin A

Summary A technique is described in which carbohydrates present in sections of animal tissues are stained with fluorescein-labelled concanavalin A. Evidence is presented which suggests that the fluorescent staining is due to the presence of hexose residues containing the -D-arabinopyranoside configuration.Possible extensions of the application of this type of histochemical method are discussed.     

Masters or slaves? Vesicle release machinery and the regulation of presynaptic calcium channels

Calcium entry through presynaptic voltage-gated calcium channels is essential for neurotransmitter release. The two major types of presynaptic calcium channels contain a synaptic protein interaction site that physically interacts with synaptic vesicle release proteins. This is thought to tighten the coupling between the sources of calcium entry and the neurotransmitter release machinery. Conversely, the binding of synaptic proteins to presynaptic calcium channels regulates calcium channel activity. Hence, presynaptic calcium channels act not only as the masters of the synaptic release process, but also as key targets for feedback inhibition.     

Effects of the binding of calcium ions on the structure and dynamics of the ΦX174 virus investigated using molecular dynamics

It is known that the presence of calcium ions (Ca^2 +) is necessary for the enterobacterial virus ΦX174 to inject its DNA into the host cell, and that some mutations in the major capsid proteins lead to better survivability at higher temperatures. Our goal in the current study is to determine the physical changes in both the wild-type and mutant virus due to the binding of Ca^2 +. Thus, we performed molecular dynamics simulations of the ΦX174 major capsid protein complex with and without Ca^2 + bound. Our results show that binding of Ca^2 + leads to energetic and dynamical changes in the virus proteins. In particular, the results suggest that binding of Ca^2 + is energetically favorable and that the mutation leads to increased fluctuations of the protein complex (especially with the calcium ions bound to the complex), which may increase the rate of genome packaging and ejection for ΦX174.     

The stimulus-secretion coupling of glucose-induced insulin release. Does glycolysis control calcium transport in the B-cell?

1. The metabolism of glucose and the exchangeable Ca2+ pool were measured in rat pancreatic islets, in order to assess the possible significance of glycolysis in the process of glucose-induced insulin release. 2. At high glucose concentration (16.7 mM), glucose was metabolized at the following rate (pmol of glucose residue/h per islet +/- S.E.M.): 131 +/- 11 for glucose uptake, 129+/-8 for glucose utilization, as judged by the conversion of [5-3H]glucose into 3H2O,60+/-2 for lactate output and 25+/-2 for glucose oxidation. 3. The secretory pattern usually correlated with the metabolic data. For instance, the ability of different sugars (glucose, mannose, fructose, galactose, D-glyceraldehyde) to stimulate lactate output closely paralleled their relative insulinotropic capacity. A disparity between metabolic and secretory responses was, however, encountered in the presence of dibutyryl cyclic AMP and theophylline. 4. Despite this contrasting behaviour, the size of the Ca2+- exchangeable pool (net uptake of 45Ca2+) was invariably proportional to the rate of lactate output under all experimental conditions examined. It is concluded that glycolysis usually exerts a tight control on the rate constant for Ca2+ transport across the B-cell membrane.     

Residue-specific fluorescent probes of α-synuclein: detection of early events at the N- and C-termini during fibril assembly

In the Parkinson's disease-associated state, α-synuclein undergoes large conformational changes, forming ordered, β-sheet-containing fibrils. To unravel the role of specific residues during the fibril assembly process, we prepared single-Cys mutants in the disordered (G7C and Y136C) and proximal (V26C and L100C) fibril core sites and derivatized them with environmentally sensitive dansyl (Dns) fluorophores. Dns fluorescence exhibits residue specificity in spectroscopic properties as well as kinetic behavior; early kinetic events were revealed by probes located at positions 7 and 136 compared to those at positions 26 and 100.     

Screening of poly-β-hydroxybutyrate-producing microorganisms using Fourier transform infrared spectroscopy

An alternative method for the analysis of poly--hydroxybutyrate (PHB) in microbial biomass based on FTIR spectroscopy was developed in which a semi-transparent, thin pellet of biomass was made with KBr and scanned by IR spectroscopy, from 400 cm^–1to 2000 cm^–1. The IR spectra of the target biomass showed a distinct absorbance band of the carboxylic ester group at 1724.03 cm^–1. The technique can be used to screen large numbers of microorganisms for PHB production.     

Fluorimetric detection of the earliest events in amyloid β oligomerization and its inhibition by pharmacologically active liposomes

BackgroundAmyloid β (Aβ) peptide aggregation is the main molecular mechanism underlying the development of Alzheimer's disease, the most widespread form of senile dementia worldwide. Increasing evidence suggests that the key factor leading to impaired neuronal function is accumulation of water-soluble Aβ oligomers rather than formation of the senile plaques created by the deposition of large fibrillary aggregates of Aβ. However, several questions remain about the preliminary steps and the progression of Aβ oligomerization.MethodsWe show that the initial stages of the aggregation of fluorescently labeled Aβ can be determined with a high degree of precision and at physiological (i.e., nanomolar) concentrations by using either steady-state fluorimetry or time-correlated single-photon counting.ResultsWe study the dependence of the oligomerization extent and rate on the Aβ concentration. We determine the chemical binding affinity of fluorescently labeled Aβ for liposomes that have been recently shown to be pharmacologically active in vivo, reducing the Aβ burden within the brain. We also probe their capacity to hinder the Aβ oligomerization process in vitro.ConclusionsWe introduced a fluorescence assay allowing investigation of the earliest steps of Aβ oligomerization, the peptide involved in Alzheimer's disease. The assay proved to be sensitive even at Aβ concentrations as low as those physiologically observed in the cerebrospinal fluid.General significanceThis work represents an extensive and quantitative study on the initial events of Aβ oligomerization at physiological concentration. It may enhance our comprehension of the molecular mechanisms leading to Alzheimer's disease, thus paving the way to novel therapeutic strategies.     

Do calcium buffers always slow down the propagation of calcium waves?

Calcium buffers are large proteins that act as binding sites for free cytosolic calcium. Since a large fraction of cytosolic calcium is bound to calcium buffers, calcium waves are widely observed under the condition that free cytosolic calcium is heavily buffered. In addition, all physiological buffered excitable systems contain multiple buffers with different affinities. It is thus important to understand the properties of waves in excitable systems with the inclusion of buffers. There is an ongoing controversy about whether or not the addition of calcium buffers into the system always slows down the propagation of calcium waves. To solve this controversy, we incorporate the buffering effect into the generic excitable system, the FitzHugh–Nagumo model, to get the buffered FitzHugh–Nagumo model, and then to study the effect of the added buffer with large diffusivity on traveling waves of such a model in one spatial dimension. We can find a critical dissociation constant ( $K=K(a)$ ) characterized by system excitability parameter $a$ such that calcium buffers can be classified into two types: weak buffers ( $K\in (K(a),\infty )$ ) and strong buffers ( $K\in (0,K(a))$ ). We analytically show that the addition of weak buffers or strong buffers but with its total concentration $b_0^1$ below some critical total concentration $b_{0,c}^1$ into the system can generate a traveling wave of the resulting system which propagates faster than that of the origin system, provided that the diffusivity $D_1$ of the added buffers is sufficiently large. Further, the magnitude of the wave speed of traveling waves of the resulting system is proportional to $\sqrt{D_1}$ as $D_1\rightarrow \infty $ . In contrast, the addition of strong buffers with the total concentration $b_0^1>b_{0,c}^1$ into the system may not be able to support the formation of a biologically acceptable wave provided that the diffusivity $D_1$ of the added buffers is sufficiently large.     

Evaluation of fasting state-/oral glucose tolerance test-derived measures of insulin release for the detection of genetically impaired β-cell function

Background

To date, fasting state- and different oral glucose tolerance test (OGTT)-derived measures are used to estimate insulin release with reasonable effort in large human cohorts required, e.g., for genetic studies. Here, we evaluated twelve common (or recently introduced) fasting state-/OGTT-derived indices for their suitability to detect genetically determined β-cell dysfunction.

Methodology/Principal Findings

A cohort of 1364 White European individuals at increased risk for type 2 diabetes was characterized by OGTT with glucose, insulin, and C-peptide measurements and genotyped for single nucleotide polymorphisms (SNPs) known to affect glucose- and incretin-stimulated insulin secretion. One fasting state- and eleven OGTT-derived indices were calculated and statistically evaluated. After adjustment for confounding variables, all tested SNPs were significantly associated with at least two insulin secretion measures (p≤0.05). The indices were ranked according to their associations'' statistical power, and the ranks an index obtained for its associations with all the tested SNPs (or a subset) were summed up resulting in a final ranking. This approach revealed area under the curve (AUC)_{Insulin(0-30)}/AUC_{Glucose(0-30)} as the best-ranked index to detect SNP-dependent differences in insulin release. Moreover, AUC_{Insulin(0-30)}/AUC_{Glucose(0-30)}, corrected insulin response (CIR), AUC_{C-Peptide(0-30)}/AUC_{Glucose(0-30)}, AUC_{C-Peptide(0-120)}/AUC_{Glucose(0-120)}, two different formulas for the incremental insulin response from 0–30 min, i.e., the insulinogenic indices (IGI)₂ and IGI₁, and insulin 30 min were significantly higher-ranked than homeostasis model assessment of β-cell function (HOMA-B; p<0.05). AUC_{C-Peptide(0-120)}/AUC_{Glucose(0-120)} was best-ranked for the detection of SNPs involved in incretin-stimulated insulin secretion. In all analyses, HOMA-β displayed the highest rank sums and, thus, scored last.

Conclusions/Significance

With AUC_{Insulin(0-30)}/AUC_{Glucose(0-30),} CIR, AUC_{C-Peptide(0-30)}/AUC_{Glucose(0-30)}, AUC_{C-Peptide(0-120)}/AUC_{Glucose(0-120)}, IGI₂, IGI₁, and insulin 30 min, dynamic measures of insulin secretion based on early insulin and C-peptide responses to oral glucose represent measures which are more appropriate to assess genetically determined β-cell dysfunction than fasting measures, i.e., HOMA-B. Genes predominantly influencing the incretin axis may possibly be best detected by AUC_{C-Peptide(0-120)}/AUC_{Glucose(0-120)}.     

Putting out the fire: what terminates calcium-induced calcium release in cardiac muscle?

The majority of contractile calcium in cardiac muscle is released from stores in the sarcoplasmic reticulum (SR), by a process of calcium-induced calcium release (CICR) through ryanodine receptors. Because CICR is intrinsically self-reinforcing, the stability of and graded regulation of cardiac EC coupling appear paradoxical. It is now well established that this gradation results from the stochastic recruitment of varying numbers of elementary local release events, which may themselves be regenerative, and which can be directly observed as calcium sparks. Ryanodine receptors (RyRs) are clustered in dense lattices, and most calcium sparks are now believed to involve activation of multiple RyRs. This implies that local CICR is regenerative, requiring a mechanism to terminate it. It was initially assumed that this mechanism was inactivation of the RyR, but during the decade since the discovery of sparks, no sufficiently strong inactivation mechanism has been demonstrated in vitro and all empirically determined gating schemes for the RyR give unstable EC coupling in Monte Carlo simulations. We consider here possible release termination mechanisms. Stochastic attrition is the spontaneous decay of active clusters due to random channel closure; calculations show that it is much too slow unless assisted by another process. Calcium-dependent RyR inactivation involving third-party proteins remains a viable but speculative mechanism; current candidates include calmodulin and sorcin. Local depletion of SR release terminal calcium could terminate release, however calculations and measurements leave it uncertain whether a sufficient diffusion resistance exists within the SR to sustain such depletion. Depletion could be assisted by dependence of RyR activity on SR lumenal [Ca(2+)]. There is substantial evidence for such lumenal activation, but it is not clear if it is a strong enough effect to account for the robust termination of sparks. The existence of direct interactions among clustered RyRs might account for the discrepancy between the inactivation properties of isolated RyRs and intact clusters. Such coupled gating remains controversial. Determining the mechanism of release termination is the outstanding unsolved problem of cardiac EC coupling, and will probably require extensive genetic manipulation of the EC coupling apparatus in its native environment to unravel the solution.     

Entropic Profiler – detection of conservation in genomes using information theory

Background

In the last decades, with the successive availability of whole genome sequences, many research efforts have been made to mathematically model DNA. Entropic Profiles (EP) were proposed recently as a new measure of continuous entropy of genome sequences. EP represent local information plots related to DNA randomness and are based on information theory and statistical concepts. They express the weighed relative abundance of motifs for each position in genomes. Their study is very relevant because under or over-representation segments are often associated with significant biological meaning.

Findings

The Entropic Profiler application here presented is a new tool designed to detect and extract under and over-represented DNA segments in genomes by using EP. It allows its computation in a very efficient way by recurring to improved algorithms and data structures, which include modified suffix trees. Available through a web interface http://kdbio.inesc-id.pt/software/ep/ and as downloadable source code, it allows to study positions and to search for motifs inside the whole sequence or within a specified range. DNA sequences can be entered from different sources, including FASTA files, pre-loaded examples or resuming a previously saved work. Besides the EP value plots, p-values and z-scores for each motif are also computed, along with the Chaos Game Representation of the sequence.

Conclusion

EP are directly related with the statistical significance of motifs and can be considered as a new method to extract and classify significant regions in genomes and estimate local scales in DNA. The present implementation establishes an efficient and useful tool for whole genome analysis.

     

Lack of effects of calcium · calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium · calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca²⁺ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium · calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca²⁺ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca²⁺ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium · calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca²⁺ release and ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange.     

Application of the pCloDF13 bacteriocin release protein in the release of heterologous proteins by Escherichia coli : Production of plant α-galactosidase

Summary The bacteriocin release protein (BRP) mediated release of several eukaryotic proteins was studied in Escherichia coli. The genes for the plant proteins -galactosidase and thaumatin, and for the mammalian proteins prochymosine and prophospholipase A₂, were cloned behind the OmpA signal peptide. The -galactosidase was expressed and secreted into the periplasm. Prochymosine and thaumatin were poorly expressed. Release experiments showed that about 5 mg/l of -galactosidase was excreted into the extracellular medium upon induction of the BRP.     

Decreased calcium channel currents and facilitated epinephrine release in the Ca channel β3 subunit-null mice

The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. The β3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in β3-null mice to determine the role of the β3 subunit in catecholamine release from the adrenal chromaffin system.Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The β3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in β3-null mice, while patch-clamp recordings showed a significant reduction in Ca²⁺-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production.In conclusion, our results indicate the importance of the β3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells.     

Sensitive detection of protein-lipid interaction change on bacteriorhodopsin using dodecyl β-D-maltoside

A light-driven proton pump bacteriorhodopsin (bR) forms a two-dimensional hexagonal lattice with about 10 archaeal lipids per monomer bR on purple membrane (PM) of Halobacterium salinarum. In this study, we found that the weakening of the bR-lipid interaction on PM by addition of alcohol can be detected as the significant increase of protein solubility in a nonionic detergent, dodecyl β-D-maltoside (DDM). The protein solubility in DDM was also increased by bR-lipid interaction change accompanied by structural change of the apoprotein after retinal removal and was about 7 times higher in the case of completely bleached membrane than that of intact PM. Interestingly, the cyclic and milliseconds order of structural change of bR under light irradiation also led to increasing the protein solubility and had a characteristic light intensity dependence with a phase transition. These results indicate that there is a photointermediate in which bR-lipid interaction has been changed by its dynamic structural change. Because partial delipidation of PM by CHAPS gave minor influence for the change of the protein solubility compared to intact PM in both dark and light conditions, it is suggested that specific interactions of bR with some lipids which remain on PM even after delipidation treatment have a key role for the change of solubility in DDM induced by alcohol binding, ligand release, and photon absorption on bR.     

Do mental events cause neural events analogously to the probability fields of quantum mechanics?

If non-material mental events, such as the intention to carry out an action, are to have an effective action on neural events in the brain, it has to be at the most subtle and plastic level of these events. In the first stage of our enquiry an introduction to conventional synaptic theory leads on to an account of the manner of operation of the ultimate synaptic units. These units are the synaptic boutons that, when excited by an all-or-nothing nerve impulse, deliver the total contents of a single synaptic vesicle, not regularly, but probabilistically. This quantal emission of the synaptic transmitter molecules (about 5000-10 000) is the elementary unit of the transmission process from one neuron to another. In the second stage this refined physiological analysis leads on to an account of the ultrastructure of the synapse, which gives clues as to the manner of its unitary probabilistic operation. The essential feature is that the effective structure of each bouton is a paracrystalline presynaptic vesicular grid with about 50 vesicles, which acts probabilistically in vesicular (quantal) release. In the third stage it is considered how a non-material mental event, such as an intention to move, could influence the subtle probabilistic operations of synaptic boutons. On the biological side, attention is focused on the paracrystalline presynaptic vesicular grids as the targets for non-material mental events. On the physical side, attention is focused on the probabilistic fields of quantum mechanics which carry neither mass nor energy, but which nevertheless can exert effective action at microsites. The new light on the mind-brain problem came from the hypothesis that the non-material mental events, the 'World 2' of Popper, relate to the neural events of the brain (the 'World 1' of matter and energy) by actions in conformity with quantum theory. This hypothesis that mental events act on probabilistic synaptic events in a manner analogous to the probability fields of quantum mechanics seems to open up an immense field of scientific investigation both in quantum physics and in neuroscience.     

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-β-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the μ-opioid receptor antagonist β-funaltrexamine and the μ₁-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the μ₁-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH₂ (d-Pro²-endomorphin-2) but not by co-administration of the μ₂-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH₂ (d-Pro²-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH₂ (d-Pro²-Tyr-W-MIF-1). In CXBK mice whose μ₁-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal μ₁-opioid receptors.