Analysis of the extreme diversity of salivary alpha-amylase isoforms generated by physiological proteolysis using liquid chromatography-tandem mass spectrometry

Saliva is a crucial biofluid for oral health and is also of increasing importance as a non-invasive source of disease biomarkers. Salivary alpha-amylase is an abundant protein in saliva, and changes in amylase expression have been previously associated with a variety of diseases and conditions. Salivary alpha-amylase is subject to a high diversity of post-translational modifications, including physiological proteolysis in the oral cavity. Here we developed methodology for rapid sample preparation and non-targeted LC-ESI-MS/MS analysis of saliva from healthy subjects and observed an extreme diversity of alpha-amylase proteolytic isoforms. Our results emphasize the importance of consideration of post-translational events such as proteolysis in proteomic studies, biomarker discovery and validation, particularly in saliva. (C) 2012 Elsevier B.V. All rights reserved.

Potential-dependent surface-enhanced resonance raman spectroscopy at nanostructured TiO2 : A case study on cytochrome b5

Abstract: Nanostructured titanium dioxide (TiO2) electrodes, prepared by anodization of titanium, are employed to probe the electron-transfer process of cytochrome b5 (cyt b5) by surface-enhanced resonance Raman (SERR) spectroscopy. Concomitant with the increased nanoscopic surface roughness of TiO2, achieved by raising the anodization voltage from 10 to 20 V, the enhancement factor increases from 2.4 to 8.6, which is rationalized by calculations of the electric field enhancement. Cyt b 5 is immobilized on TiO2 under preservation of its native structure but it displays a non-ideal redox behavior due to the limited conductivity of the electrode material. The electron-transfer efficiency which depends on the crystalline phase of TiO2 has to be improved by appropriate doping for applications in bioelectrochemistry. Nanostructured TiO2 electrodes are employed to probe the electron-transfer process of cytochrome b5 by surface-enhanced resonance Raman spectroscopy. Concomitant with the increased nanoscopic surface roughness of TiO2, the enhancement factor increases, which can be attributed to the electric field enhancement. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Amino group positions dependent morphology and coverage of electropolymerized metallophthalocyanine (M = Ni and Co) films on electrode surfaces

Electropolymerized films of teraaminometallophthalocyanines (MTAPc; M = Ni and Co) with amino groups at α- (4α-MTAPc) and β- (4β-MTAPc) positions were prepared on glassy carbon (GC) and indium tin oxide (ITO) electrodes. It was found that the electropolymerization growth rate of 4α-MTAPc was less than that of 4β-MTAPc prepared under identical conditions. Further, the surface coverage of the polymerized 4β-MTAPc film was greater than that of 4α-MTAPc polymerized film. Atomic force microscopy (AFM), X-ray diffraction (XRD) and UV–visible spectroscopic studies were carried out for the polymerized films of 4α-NiIITAPc (p-4α-NiIITAPc) and 4β-NiIITAPc (p-4β-NiIITAPc) alone because both Ni(II) and Co(II) polymerized films show similar trend in electropolymerization and surface coverage values. AFM images show that p-4α-NiIITAPc film contains islands and the thickness of this film was nearly three times less than that of p-4β-NiIITAPc. XRD patterns for the two polymerized films reveal that p-4β-NiIITAPc film was relatively more crystalline than p-4α-NiIITAPc film. Further, the compactness of these films was scrutinized from their barrier properties toward [Fe(CN)6]3−/4− redox couple. The differences in the polymerization growth rate of 4α-MTAPc and 4β-MTAPc, and the thicknesses of the resultant polymerized films suggest that unlike 4β-MTAPc one or two amino groups might have not involved in electropolymerization in the case of 4α-MTAPc. Further, the influence of surface coverage on the electrocatalytic properties of the polymerized films was studied by taking p-4β-CoIITAPc and p-4α-CoIITAPc films as examples. The electrocatalytic oxygen reduction current was almost same at both the electrodes suggesting that only the surface species were involved in the electrocatalytic reduction of oxygen.

Amino group position dependent orientation of self-assembled monomolecular films of tetraaminophthalocyanatocobalt(II) on Au surfaces

Self-assembled monomolecular films of 1,8,15,22-tetraaminophthalocyanatocobalt(II) (4α-CoIITAPc) and 2,9,16,23-tetraaminophthalocyanatocobalt(II) (4β-CoIITAPc) on Au surfaces were prepared by spontaneous adsorption from solution. These films were characterized by cyclic voltammetry and Raman spectroscopy. Both the surface coverage (Γ) and intensity of the in-plane stretching bands obtained from Raman studies vary for these monomolecular films, indicating different orientations adopted by them on Au surfaces. The 4α-CoIITAPc-modified electrode exhibits an E1/2 of 0.35 V, while the 4β-CoIITAPc-modified electrode exhibits an E1/2 of 0.19 V, corresponding to the CoII/CoIII redox couple in 0.1 M H2SO4. The Γ estimated from the charge associated with the oxidation of Co(II) gives (2.62 ± 0.10) × 10-11 mol cm-2 for 4α-CoIITAPc and (3.43 ± 0.14) × 10-10 mol cm-2 for 4β-CoIITAPc. In Raman spectral studies, the intensity ratio between in-plane phthalocyanine (Pc) stretching and the Au−N stretching was found to be 6.6 for 4β-CoIITAPc, while it was 1.6 for 4α-CoIITAPc. The obtained lower Γ and intensity ratio values suggest that 4α-CoIITAPc adopts nearly a parallel orientation on the Au surface, while the higher Γ and intensity ratio values suggest that 4β-CoIITAPc adopts a perpendicular orientation. The electrochemical reduction of dioxygen was carried out using these differently oriented Pc's in phosphate buffer solution (pH 7.2). Both the Pc's catalyze the reduction of dioxygen; however, the 4α-CoIITAPc-modified electrode greatly reduces the dioxygen reduction overpotential compared to 4β-CoIITAPc-modified and bare Au electrodes.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

Towards a quantitative theory of epidermal calcium profile formation in unwounded skin

We propose and mathematically examine a theory of calcium profile formation in unwounded mammalian epidermis based on: changes in keratinocyte proliferation, fluid and calcium exchange with the extracellular fluid during these cells' passage through the epidermal sublayers, and the barrier functions of both the stratum corneum and tight junctions localised in the stratum granulosum. Using this theory, we develop a mathematical model that predicts epidermal sublayer transit times, partitioning of the epidermal calcium gradient between intracellular and extracellular domains, and the permeability of the tight junction barrier to calcium ions. Comparison of our model's predictions of epidermal transit times with experimental data indicates that keratinocytes lose at least 87% of their volume during their disintegration to become corneocytes. Intracellular calcium is suggested as the main contributor to the epidermal calcium gradient, with its distribution actively regulated by a phenotypic switch in calcium exchange between keratinocytes and extracellular fluid present at the boundary between the stratum spinosum and the stratum granulosum. Formation of the extracellular calcium distribution, which rises in concentration through the stratum granulosum towards the skin surface, is attributed to a tight junction barrier in this sublayer possessing permeability to calcium ions that is less than 15 nm/s in human epidermis and less than 37 nm/s in murine epidermis. Future experimental work may refine the presented theory and reduce the mathematical uncertainty present in the model predictions.

Characterization of frequency‐dependent magnetic susceptibility in UXO electromagnetic geophysics

Soils at many locations that have their origin in volcanic parent material and have undergone extensive weathering often exhibit strong frequency-dependent magnetic susceptibilities. The presence of such susceptibility has a profound effect on electromagnetic induction data acquired in such environments. Their transient electromagnetic response is characterized by a t-1 decay that is strong enough to mask UXO responses. In a field study and associated laboratory work on characterizing the frequency-dependent magnetic susceptibility and its influence on transient electromagnetic data, we collected soil samples on the surface and in soil pits from the Island of Kaho'olawe, Hawaii, and measured their frequency dependent magnetic susceptibilities. We present the details of the field investigation, confirm previous theoretical work with field and laboratory measurements, characterize the susceptibility with a Cole-Cole model, and investigate the response specific to the measured susceptibility.

Stress at the synapse : signal transduction mechanisms of adrenal steroids at neuronal membranes

As the key neuron-to-neuron interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. However, the signal transduction mechanisms by which stress mediates its lasting effects on synapse transmission and on memory are not fully understood. A key component of the stress response is the increased secretion of adrenal steroids. Adrenal steroids (e.g., cortisol) bind to genomic mineralocorticoid and glucocorticoid receptors (gMRs and gGRs) in the cytosol. In addition, they may act through membrane receptors (mMRs and mGRs), and signal transduction through these receptors may allow for rapid modulation of synaptic transmission as well as modulation of membrane ion currents. mMRs increase synaptic and neuronal excitability; mechanisms include the facilitation of glutamate release through extracellular signal-regulated kinase signal transduction. In contrast, mGRs decrease synaptic and neuronal excitability by reducing calcium currents through N-methyl-D-aspartate receptors and voltage-gated calcium channels by way of protein kinase A- and G protein-dependent mechanisms. This body of functional data complements anatomical evidence localizing GRs to the postsynaptic membrane. Finally, accumulating data also suggest the possibility that mMRs and mGRs may show an inverted U-shaped dose response, whereby glutamatergic synaptic transmission is increased by low doses of corticosterone acting at mMRs and decreased by higher doses acting at mGRs. Thus, synaptic transmission is regulated by mMRs and mGRs, and part of the stress signaling response is a direct and bidirectional modulation of the synapse itself by adrenal steroids.

Mathematical models of calcium and tight junctions in normal and reconstructed epidermis

This project investigated the calcium distributions of the skin, and the growth patterns of skin substitutes grown in the laboratory, using mathematical models. The research found that the calcium distribution in the upper layer of the skin is controlled by three different mechanisms, not one as previously thought. The research also suggests that tight junctions, which are adhesions between neighbouring skin cells, cannot be solely responsible for the differences in the growth patterns of skin substitutes and normal skin.

Exploration of the fundamental equilibrium behaviour of calcium exchange with weak acid cation resins

This study evaluated the complexity of calcium ion exchange with sodium exchanged weak acid cation resin (DOW MAC-3). Exchange equilibria recorded for a range of different solution normalities revealed profiles which were represented by conventional “L” or “H” type isotherms at low values of equilibrium concentration (Ce) of calcium ions, plus a superimposed region of increasing calcium uptake was observed at high Ce values. The loading of calcium ions was determined to be ca. 53.5 to 58.7 g/kg of resin when modelling only the sorption curve created at low Ce values,which exhibited a well-defined plateau. The calculated calcium ion loading capacity for DOWMAC-3 resin appeared to correlate with the manufacturer's recommendation. The phenomenon of super equivalent ion exchange (SEIX) was observed when the “driving force” for the exchange process was increased in excess of 2.25 mmol calcium ions per gram of resin in the starting solution. This latter event was explained in terms of displacement of sodium ions from sodium hydroxide solution which remained in the resin bead following the initial conversion of the as supplied “H+” exchanged resin sites to the “Na+” version required for softening studies. Evidence for hydrolysis of a small fraction of the sites on the sodium exchanged resin surface was noted. The importance of carefully choosing experimental parameters was discussed especially in relation to application of the Langmuir–Vageler expression. This latter model which compared the ratio of the initial calcium ion concentration in solution to resin mass, versus final equilibrium loading of the calcium ions on the resin; was discovered to be an excellent means of identifying the progress of the calcium–sodium ion exchange process. Moreover, the Langmuir–Vageler model facilitated standardization of various calcium–sodium ion exchange experiments which allowed systematic experimental design.

Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders. In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed. In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.

Experimental and numerical investigation of strain-rate dependent mechanical properties of single living cells

The objective of this project is to investigate the strain-rate dependent mechanical behaviour of single living cells using both experimental and numerical techniques. The results revealed that living cells behave as porohyperlastic materials and that both solid and fluid phases within the cells play important roles in their mechanical responses. The research reported in this thesis provides a better understanding of the mechanisms underlying the cellular responses to external mechanical loadings and of the process of mechanical signal transduction in living cells. It would help us to enhance knowledge of and insight into the role of mechanical forces in supporting tissue regeneration or degeneration.

Estimating link-dependent origin-destination matrices from sample trajectories and traffic counts

In transport networks, Origin-Destination matrices (ODM) are classically estimated from road traffic counts whereas recent technologies grant also access to sample car trajectories. One example is the deployment in cities of Bluetooth scanners that measure the trajectories of Bluetooth equipped cars. Exploiting such sample trajectory information, the classical ODM estimation problem is here extended into a link-dependent ODM (LODM) one. This much larger size estimation problem is formulated here in a variational form as an inverse problem. We develop a convex optimization resolution algorithm that incorporates network constraints. We study the result of the proposed algorithm on simulated network traffic.

Amygdala-dependent learning increases the number of spinophilin-immunoreactive dendritic spines in the lateral amygdala

During Pavlovian auditory fear conditioning a previously neutral auditory stimulus (CS) gains emotional significance through pairing with a noxious unconditioned stimulus (US). These associations are believed to be formed by way of plasticity at auditory input synapses on principal neurons in the lateral nucleus of the amygdala (LA). One proposed form of cellular plasticity involves structural changes in the number and morphology of dendritic spines...