The continuing disappearance of "pure” Ca2+buffers

Abstract.: Advances in the understanding of a class of Ca2+-binding proteins usually referred to as "Ca2+buffers” are reported. Proteins historically embraced within this group include parvalbumins (α and β), calbindin-D9k, calbindin-D28k and calretinin. Within the last few years a wealth of data has accumulated that allow a better understanding of the functions of particular family members of the >240 identified EF-hand Ca2+-binding proteins encoded by the human genome. Studies often involving transgenic animal models have revealed that they exert their specific functions within an intricate network consisting of many proteins and cellular mechanisms involved in Ca2+ signaling and Ca2+ homeostasis, and are thus an essential part of the Ca2+ homeostasome. Recent results indicate that calbindin-D28k, possibly also calretinin and oncomodulin, the mammalian β parvalbumin, might have additional Ca2+ sensor functions, leaving parvalbumin and calbindin-D9k as the only "pure” Ca2+buffer

Laser-induced chemical liquid-phase deposition of copper on transparent substrates

Laser-induced chemical liquid phase deposition allows maskless manufacturing of metallic structures on the surface of dielectrics and is prospected to be a promising tool in the field of microelectronics and microfluidics. The aim of the work presented here is to combine this deposition method with a related micro-structuring method known as laser-induced backside wet etching. Fabricating both, microstructured surface structures and subsequent deposition of conducting patterns within the same setup would be an interesting tool for rapid prototyping.To demonstrate the functional principle of this combined approach conductive copper lines were deposited at the backside of both polished and structured soda lime glass substrates by using a focused, scanning ns-pulsed Ytterbium fiber laser at 532nm wavelength. The deposition process is initiated by a photo induced reaction of a CuSO4-based liquid precursor in contact with the backside of the substrate. The obtained metallic copper deposits are crystalline, stable under ambient conditions and have a conductivity in the same order of magnitude as bulk copper

Influence of the pre-treatment of glass substrates on Laser-Induced Backside Wet Etching using NIR Nanosecond-Pulses and Cu-based solutions

Laser induced backside wet etching (LIBWE) has shown to be a promising tool for the micro-structuring of transparent materials. Our group has investigated LIBWE using NIR ns-laser pulses and Cu-based absorber liquids. Focus of this paper is to investigate the influence of the pre-treatment of the transparent substrate on ablation. For this purpose experiments were done on untreated and silanized soda lime glass surfaces. Our results show that depending on the absorber liquid the silanization of the substrate either enhances or delays the ablation. Possible ablation models for the different experimental settings will be discussed

Restricted diffusion of calretinin in cerebellar granule cell dendrites implies Ca²⁺-dependent interactions via its EF-hand 5 domain

Ca²⁺-binding proteins (CaBPs) are important regulators of neuronal Ca²⁺ signaling, acting either as buffers that shape Ca²⁺ transients and Ca²⁺ diffusion and/or as Ca²⁺ sensors. The diffusional mobility represents a crucial functional parameter of CaBPs, describing their range-of-action and possible interactions with binding partners. Calretinin (CR) is a CaBP widely expressed in the nervous system with strong expression in cerebellar granule cells. It is involved in regulating excitability and synaptic transmission of granule cells, and its absence leads to impaired motor control. We quantified the diffusional mobility of dye-labelled CR in mouse granule cells using two-photon fluorescence recovery after photobleaching (FRAP). We found that movement of macromolecules in granule cell dendrites was not well described by free Brownian diffusion and that CR diffused unexpectedly slow compared to fluorescein dextrans of comparable size. During bursts of action potentials, which were associated with dendritic Ca²⁺ transients, the mobility of CR was further reduced. Diffusion was significantly accelerated by a peptide embracing EF-hand 5 of CR. Our results suggest long-lasting, Ca²⁺-dependent interactions of CR with large and/or immobile binding partners. These interactions render CR a poorly mobile Ca²⁺ buffer and point towards a Ca²⁺ sensor function of CR

Upregulated expression of oncomodulin, the beta isoform of parvalbumin, in perikarya and axons in the diencephalon of parvalbumin knockout mice

The calcium-binding proteins parvalbumin, calbindin D-28k, calretinin and calcineurin are present in subsets of GABAergic gigantic calyciform presynaptic terminals of the reticular thalamic nucleus (RTN). Previously it was hypothesized that GABA and calcium-binding proteins including parvalbumin are not only colocalized in the same neuron subpopulation, but that GABA synthesis and parvalbumin expression could be also genetically regulated by a common mechanism. Moreover, parvalbumin expression levels could influence GABA synthesis. For this, we analyzed GABA immunoreactivity in RTN gigantic calyciform presynaptic terminals of parvalbumin–deficient (PV−/−) mice. With respect to GABA immunoreactivity we found no differences compared to wild–type animals. However, using a polyclonal parvalbumin antibody raised against full-length rat muscle parvalbumin on brain sections of PV−/− mice, we observed paradoxical parvalbumin immunoreactivity in partly varicose axons in the diencephalon, mainly in the lamina medullaris externa surrounding the thalamus. A detailed immunohistochemical, biochemical and molecular biological analysis revealed this immunoreactivity to be the result of an upregulation of oncomodulin (OM), the mammalian beta isoform of parvalbumin in PV−/− mice. In addition, OM was present in a sparse subpopulation of neurons in the thalamus and in the dentate gyrus. OM expression has not been observed before in neurons of the mammalian brain; its expression was restricted to outer hair cells in the organ of Corti. Our results indicate that the absence of parvalbumin has no major effect on the GABA-synthesizing system in RTN presynaptic terminals excluding a direct effect of parvalbumin on this regulation. However, a likely homeostatic mechanism is induced resulting in the upregulation of OM in selected axons and neuronal perikarya. Our results warrant further detailed investigations on the putative role of OM in the brain

Measuring Optical Properties On Rough And Liquid Metal Surfaces

For understanding and optimizing laser processing of metals and alloys the optical properties, especially the absorption of the work piece in function of the temperature up to the liquid phase have to be known [1]. There are several approaches to extend the Drude-Model [2] for optical properties of metal to temperature dependence [3, 4, 5]. However, a verification of these models is difficult due to the lack of sufficient experimental data. Even though measuring optical properties with ellipsometry is well established, such measurements on metals and alloys at elevated temperatures up to the liquid state are very challenging. To collect the optical properties of different metals and alloys like Al, Ti, Ag, Cu and steel in the solid and liquid state a custom-made high-temperature ellipsometer was used. The instrument is also used to investigate the influence of curved and rough surfaces which may occur due to the heating of the samples during the ellipsometric measurements

Development of an enzyme-linked immunosorbent assay for the detection of human calretinin in plasma and serum of mesothelioma patients

<p>Abstract</p> <p>Background</p> <p>Calretinin is one of the well-established immunohistochemical markers in the diagnostics of malignant mesothelioma (MM). Its utility as a diagnostic tool in human blood, however, is scarcely investigated. The aim of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for human calretinin in blood and to assess its usefulness as a potential minimally invasive diagnostic marker for MM.</p> <p>Methods</p> <p>Initially, attempts were made to establish an assay using commercially available antibodies and to optimize it by including a biotin-streptavidin complex into the assay protocol. Subsequently, a novel ELISA based on polyclonal antibodies raised in rabbit immunized with human recombinant calretinin was developed. The assay performance in human serum and plasma (EDTA/heparin) and the influence of calcium concentrations on antibody recognition were studied. Stability of spiked-in calretinin in EDTA plasma under different storage conditions was also examined. In preliminary studies serum and plasma samples from 97 healthy volunteers, 35 asbestos-exposed workers, and 42 MM patients were analyzed.</p> <p>Results</p> <p>The mean detection range of the new ELISA was 0.12 to 8.97 ng/ml calretinin. The assay demonstrated markedly lower background and significantly higher sensitivity compared to the initially contrived assay that used commercial antibodies. Recovery rate experiments confirmed dependence of calretinin antibody recognition on calcium concentration. Calcium adjustment is necessary for calretinin measurement in EDTA plasma. Spiked-in calretinin revealed high stability in EDTA plasma when stored at room temperature, 4°C, or after repeated freeze/thaw cycles. Median calretinin values in healthy volunteers, asbestos workers, and MM patients were 0.20, 0.33, and 0.84 ng/ml, respectively (p < 0.0001 for healthy vs. MM, p = 0.0036 for healthy vs. asbestos-exposed, p < 0.0001 for asbestos-exposed vs. MM). Median values in patients with epithelioid and biphasic MM were similar. No influence of age, gender, smoking status, or type of medium (plasma/serum) on calretinin values was found.</p> <p>Conclusions</p> <p>The novel assay is highly sensitive and applicable to human serum and plasma. Calretinin appears to be a promising marker for the blood-based detection of MM and might complement other markers. However, further studies are required to prove its usefulness in the diagnosis of MM patients.</p

Comparison of Disdrometer and Rain Gauge Measurements During Pre-CHUVA

Cloud processes of the main precipitation systems in Brazil: A contribution to cloud resolving modeling and to the global precipitation measurement

Posttranscriptional regulation controls calretinin expression in malignant pleural mesothelioma

Calretinin (CALB2) is a diagnostic and prognostic marker in malignant pleural mesothelioma (MPM). We previously reported that calretinin expression is regulated at the mRNA level. The presence of a medium-sized (573 nucleotide) 3′ untranslated region (3′UTR) predicted to contain binding sites for miR-30a/b/c/d/e and miR-9 as well as an adenine/uridine-rich element (ARE) in all three transcripts arising from the CALB2 gene, suggests that calretinin expression is regulated via posttranscriptional mechanisms. Our aim was to investigate the role of the CALB2-3′UTR in the posttranscriptional regulation of calretinin expression in MPM. CALB2-3′UTR was inserted downstream of the luciferase reporter gene using pmiRGLO vector and reporter expression was determined after transfection into MPM cells. Targeted mutagenesis was used to generate variants harboring mutated miR-30 family and ARE binding sites. Electrophoretic mobility shift assay was used to test for the presence of ARE binding proteins. CALB2-3′UTR significantly decreased luciferase activity in MPM cells. Analysis of mutation in the ARE site revealed a further destabilization of the reporter and human antigen R (HuR) binding to the ARE sequence was detected. The mutation of two miR-30 binding sites abolished CALB2-3′UTR destabilization effect; a transient delivery of miR-30e-5p mimics or anti-miR into MPM cells resulted in a significant decrease/increase of the luciferase reporter expression and calretinin protein, respectively. Moreover, overexpression of CALB2-3′UTR quenched the effect of miR-30e-5p mimics on calretinin protein levels, possibly by sequestering the mimics, thereby suggesting a competitive endogenous RNA network. Finally, by data mining we observed that expression of miR-30e-5p was negatively correlated with the calretinin expression in a cohort of MPM patient samples. Our data show the role of (1) adenine-uridine (AU)-binding proteins in calretinin stabilization and (2) miR-30e-5p in the posttranscriptional negative regulation of calretinin expression via interaction with its 3′UTR. Furthermore, our study demonstrates a possible physiological role of calretinin’s alternatively spliced transcripts