100% efficient sub-nanoliter sample introduction in laser-induced breakdown spectroscopy and inductively coupled plasma spectrometry: implications for ultralow sample volumes.

Recently, nanoflow nebulizers with low-volume drain-free spray chambers became available for inductively coupled plasma-mass spectrometry application for analysis of very small sampling volumes. The present technical note reports on a different approach for 100% efficient subnanoliter sample introduction, the application of monodisperse piezoelectric microdroplet dispensers which generate 40-50 microm droplets with high reproducibility if nozzles of 30 microm diameter are applied. The droplets having volumes below 0.1 nL can be introduced loss-free and without plasma loading, with frequencies up to approximately 100 Hz into analytical plasmas. In this technical note, the analytical figures of merit of laser-induced breakdown spectroscopy and inductively coupled plasma-optical emission spectrometry with single droplet introduction are reported using Ca and Au standard solutions as examples. Future engineering is required to reduce the total sample volumes from the relatively large sample reservoir of the current study, thereby reducing potential issues of washout while enabling analysis of ultralow total sample volumes.

Common pathological mechanisms in mouse models for muscular dystrophies.

Duchenne/Becker and limb-girdle muscular dystrophies share clinical symptoms like muscle weakness and wasting but differ in clinical presentation and severity. To get a closer view on the differentiating molecular events responsible for the muscular dystrophies, we have carried out a comparative gene expression profiling of hindlimb muscles of the following mouse models: dystrophin-deficient (mdx, mdx(3cv)), sarcoglycan-deficient (Sgca null, Sgcb null, Sgcg null, Sgcd null), dysferlin-deficient (Dysf null, SJL(Dysf)), sarcospan-deficient (Sspn null), and wild-type (C57Bl/6, C57Bl/10) mice. The expression profiles clearly discriminated between severely affected (dystrophinopathies and sarcoglycanopathies) and mildly or nonaffected models (dysferlinopathies, sarcospan-deficiency, wild-type). Dystrophin-deficient and sarcoglycan-deficient profiles were remarkably similar, sharing inflammatory and structural remodeling processes. These processes were also ongoing in dysferlin-deficient animals, albeit at lower levels, in agreement with the later age of onset of this muscular dystrophy. The inflammatory proteins Spp1 and S100a9 were up-regulated in all models, including sarcospan-deficient mice, which points, for the first time, at a subtle phenotype for Sspn null mice. In conclusion, we identified biomarker genes for which expression correlates with the severity of the disease, which can be used for monitoring disease progression. This comparative study is an integrating step toward the development of an expression profiling-based diagnostic approach for muscular dystrophies in humans.

Flight simulator evaluation of a novel flight instrument display to minimize the risks of spatial disorientation.

BACKGROUND: Spatial disorientation (SD) in flight remains a major source of attrition. Many SD accidents would occur regardless of the instrument display in use, since the aircrew are simply not looking at the instruments. However, there are a number of accidents which might be amenable to improved instrument displays. In an attempt to improve maintenance and reattainment of correct orientation with a reduced cognitive workload, a novel instrument display has been developed. This paper describes an assessment of the display in a UH-60 helicopter flight simulator. HYPOTHESIS: This study tested the hypothesis that during instrument flight and recovery from unusual attitudes, the novel display permits a more accurate maintenance and reestablishment of flight parameters than the standard flight instruments. METHODS: There were 16 male aviators who flew a simulated instrument flight profile and recovery from unusual attitudes using both the standard flight instruments and the novel display. The two display formats were tested both with and without a secondary task. RESULTS: When compared with the standard instruments, both control of flight parameters and recovery from unusual attitudes were significantly improved when using the novel display. Analysis of the secondary task scores showed that cognitive workload was reduced when using the novel display compared with the standard instruments. CONCLUSIONS: Results from all aspects of the assessment indicated benefits of the new display. Future testing should be carried out during real flight, and the display should be further developed to be used in a head-up or helmet-mounted device.

[Secretory immunoglobulin A in saliva of healthy children and children with airway diseases]. / Sekretorisches Immunglobulin A im Speichel von gesunden Kindern und Kindern mit Atemwegserkrankungen.

Using the Elisa-Test of Dakopatts, Hamburg, described by Ishiguro et al and modified by us (Mikrotitration plates instead of tubes, blocking up free bonding capacities in the plates with 1% gel fluid, altered incubation periods) we determined secretory IgA (SIgA) in saliva samples of 376 infants and children. The probands could be divided in three groups: Group 1, serving as controls, consisted of 163 healthy children. Group 2 comprised 111 children suffering from acute infection of the respiratory tract. Group 3 consisted of 102 children with chronic airways diseases, in particular, asthma. In the healthy infants and children we found age dependent increases of SIgA until the age of 4 years. The median values amounted 16.7 (newborns), 59.2 (1st year), 118.2 (2nd year), 149.2 (3rd year), 185.5 (4th year), 159 (5th year) and 175.8 mg/l (5th-13th year). A similar age dependent increase of SIgA was evident in the saliva samples of children suffering from acute infections of the respiratory tract. In the children with chronic airways diseases there was only a slight increase of SIgA during the first 4 years (mean = 78.0-113.5 mg/l) and an abrupt (statistically significant) rise in the fifth year. The median value of SIgA was 216 mg/l in the children aged 5-13 years. Serum IgA along with salivary IgA were measured in 128 children (r = 0.40, p less than 0.001). 6 children had a complete IgA deficiency and 4 children an incomplete IgA deficiency, i.e. low secretory IgA levels in saliva (36.8-50.0 mg/l) and lacking IgA in serum (less than 14 mg/dl).(ABSTRACT TRUNCATED AT 250 WORDS)

Functional interaction of the cytoplasmic domain of triadin with the skeletal ryanodine receptor.

Triadin has been shown to co-localize with the ryanodine receptor in the sarcoplasmic reticulum membrane. We show that immunoprecipitation of solubilized sarcoplasmic reticulum membrane with antibodies directed against triadin or ryanodine receptor, leads to the co-immunoprecipitation of ryanodine receptor and triadin. We then investigated the functional importance of the cytoplasmic domain of triadin (residues 1-47) in the control of Ca2+ release from sarcoplasmic reticulum. We show that antibodies directed against a synthetic peptide encompassing residues 2-17, induce a decrease in the rate of Ca2+ release from sarcoplasmic reticulum vesicles as well as a decrease in the open probability of the ryanodine receptor Ca2+ channel incorporated in lipid bilayers. Using surface plasmon resonance spectroscopy, we defined a discrete domain (residues 18-46) of the cytoplasmic part of triadin interacting with the purified ryanodine receptor. This interaction is optimal at low Ca2+ concentration (up to pCa 5) and inhibited by increasing calcium concentration (IC50 of 300 microM). The direct molecular interaction of this triadin domain with the ryanodine receptor was confirmed by overlay assay and shown to induce the inhibition of the Ca2+ channel activity of purified RyR in bilayer. We propose that this interaction plays a critical role in the control, by triadin, of the Ca2+ channel behavior of the ryanodine receptor and therefore may represent an important step in the regulation process of excitation-contraction coupling in skeletal muscle.

Cloning and characterization of a new isoform of skeletal muscle triadin.

We have shown that several isoforms of triadin, a protein involved in calcium release process through the ryanodine receptor, are expressed in rat skeletal muscle, and we have cloned two of these isoforms. One is the rat homolog of the 95-kDa triadin identified in rabbit skeletal muscle, and the second one, shorter, is a truncated form of the previous one, but with a new unique COOH-terminal end. We propose to name the two proteins identified here Trisk 95 and Trisk 51. We have produced antibodies specific to each isoform. Using these antibodies, we have shown that the newly identified protein, Trisk 51, is actually expressed in adult rat skeletal muscle and also in rat embryo skeletal muscle. Immunofluorescent labeling of rat skeletal muscle with anti-Trisk 95, anti-Trisk 51, or anti-ryanodine receptor antibodies shows a similar localization of these proteins, in the tissue. Transfection of L6 cells with cDNA of Trisk 51 or Trisk 95 leads to the expression of proteins with the expected molecular weight, identical to those detected in rat skeletal muscle. Both proteins appear during differentiation of satellite cells in myotubes which may indicate the involvement of these two isoforms in the building of a functional calcium release machinery.

Interaction of S100A1 with the Ca2+ release channel (ryanodine receptor) of skeletal muscle.

In the present report we studied the interaction between the skeletal muscle ryanodine receptor and the ubiquitous S100A1 Ca2+ binding protein. S100A1 did not affect equilibrium [3H]ryanodine binding to purified rabbit skeletal muscle terminal cisternae at 100 microM free [Ca2+]. At nanomolar free [Ca2+], however, S100A1 activated by 40 +/- 6.7% (mean +/- SE, n = 5) the [3H]ryanodine binding activity; the half-maximal concentration for stimulation of [3H]ryanodine binding was approximately 70 nM, a value well below the estimated S100A1 concentration in skeletal muscle fibers. Scatchard analysis of [3H]ryanodine binding performed in the presence of 100 microM EGTA indicates that S100A1 increases the apparent affinity of the receptor for ryanodine (Kd = 191 vs 383 nM in the presence and in the absence of 100 nM S100A1, respectively). The effect of S100A1 was also tested on the single-channel gating properties of the purified ryanodine receptor after reconstitution into a lipid planar bilayer. Currents carried by purified ryanodine receptor channels were modulated by both cis Ca2+ and ruthenium red. In the presence of nanomolar [Ca2+], S100A1 activated the channel by increasing (6.0 +/- 2.8)-fold (mean +/- SE, n = 3) the normalized open probability. The interaction between S100A1 and the purified RYR was verified using the optical biosensor BIAcore: we show that the two proteins interact directly both at millimolar and at nanomolar calcium concentrations. We next mapped the regions of the skeletal muscle RYR involved in the interaction with S100A1 by performing ligand overlays on a panel RYR of fusion proteins in the presence of 100 nM S100A1. Our results indicate that the skeletal muscle RYR contains three potential S100A1 binding domains. Binding of S100A1 to the RYR fusion proteins occurred at both nanomolar and millimolar free [Ca2+]. S100A1 binding domain 1 binds the ligand in the presence of 1 mM free [Ca2+] or 1 mM EGTA. Maximal binding to S100A1#2 was achieved in the presence of 1 mM free [Ca2+]. The S100A1#3 domain, which overlaps with calcium-dependent calmodulin binding domain 3 (CaM 3), exhibits weak and strong S100A1 binding activity in the presence of either millimolar or nanomolar Ca2+, respectively. The interaction between S100A1 and the purified RYR complex was also investigated by affinity chromatography: in the presence of nanomolar Ca2+, we observed binding of native RYR complex to S100A1-conjugated Sepharose. This interaction could be inhibited by the presence of RYR polypeptides encompassing S100A1 binding sites S100A1#1, S100A1#2, and S100A1#3.