Thiolato-bridged “Linear” trinuclear platinum complexes [Pt3(μ-SR)4(dppm)2]

Thiolato-bridged tri- and dinuclear platinum complexes of the types [Pt₃(μ-SR)₄(dppm)₂]²⁺ (1) and [Pt₂(μ-ER)₂(dppm)₂]²⁺ (2) (E=S or Se; R=alkyl or aryl; dppm=bis(diphenylphosphino)methane) have been prepared using the mononuclear precursors [Pt(ER)₂(dppm)]. The complexes have been characterized by NMR (¹H, ¹³C, ³¹P, ¹⁹⁵Pt), FT-IR and FAB mass spectral data. The structure of [Pt₃(μ-SC₆H₄CH₃-4)₄(dppm)₂][CF₃SO₃]₂ · 6CH₂Cl₂ (1d), has been established through X-ray crystallography, revealing a zig-zag arrangement of the three coordination spheres around the platinum atoms.     

Salivation into sieve elements in relation to plant chemistry: the case of the aphid Sitobion fragariae and the wheat, Triticum aestivum

Extended sieve element salivation (E1 waveform in the electrical penetration graph) is a characteristic activity during early sieve element punctures, particularly in resistant plants. In order to explore a chemically-mediated mechanism of resistance associated with sieve element salivation, we compared the pattern of feeding behaviour of the aphid, Sitobion fragariae (Walker), on two cultivars of the wheat Triticum aestivum L., with different concentrations of hydroxamic acids (Hx). During 24 h of electronic monitoring, aphids dedicated over 50% of the total time to phloem ingestion from the sieve elements. Total time allocated to E1 in the experiment, time to first E1 within the experiment, time allocated to E1 before a sustained phloem ingestion (E2) and the contribution of sieve element salivation to the phloem phase (E1/[E1+E2]) were significantly higher in the high-Hx cultivar. The increased salivation in plants with higher contents of Hx suggests the existence, at least in this system, of a chemically-mediated sieve element constraint.     

Mikrospektrofluorometrische Untersuchungen der biogenen Amine im Glomus caroticum des Kaninchens nach Reserpin- und PCPA-Applikation

Zusammenfassung Die Glomera carotica von 12 Kaninchen wurden nach viertägiger Applikation von Reserpin (2 mg/kg KG; i.p.) (I), Parachlorphenylalanin-methylester-hydrochlorid (PCPA; 100 mg/kg KG; i.p.) (II) und physiologischer NaCl-Lösung (III) mit der Falckschen Fluoreszenzmethode aufgearbeitet und cytospektrofluorometrisch (Mikrospektrograph Leitz) ausgewertet.An Hand der Fluoreszenzspektren der spezifischen Glomuszellen (Typ I) ließ sich eine Depletion der Catecholamine und des Serotonins verfolgen: hiernach senkt PCPA vor allem das Serotonin, Reserpin hingegen die Catechol- und Indolamine in etwa gleichem Umfang.Durch Zerlegung einiger Gesamtspektren in die Anteile für Catecholamine und das Serotonin mit Hilfe eines Iterationsverfahrens der nichtlinearen Regression konnte gezeigt werden, daß nur eine geringfügige Beeinflussung der Catecholamin-Peakhöhe durch das Serotonin vorliegt. Über die Messung der Peakhöhe im Catecholaminbereich der Gesamtspektren konnte daher die Reduktion der Catecholamine auf 64±22% durch PCPA und auf 13±4% durch Reserpin statistisch gesichert werden.Die Befunde werden im Zusammenhang mit den möglichen Wirkungsmechanismen der applizierten Substanzen diskutiert. Eine ausführliche tabellarische Zusammenstellung der bisherigen Angaben über Gehalt und Zusammensetzung der biogenen Amine des Glomus caroticum ergänzt diese Diskussion.

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Microspectrofluorometric investigation of biogenic amines in the rabbit glomus caroticum after reserpine and PCPA treatment

Summary After the administration for 4 days of (I) reserpine (2 mg/kg body weight daily, i.p.), (II) p-chlorophenylalanine-methylester-hydrochloride (PCPA; 100 mg/kg body weight daily; i.p.) and III physiological NaCl solution, the carotid bodies of 12 rabbits were examined using Falck's fluorescence technique and evaluated cytospectrofluorometrically. Fluorescence spectra of the type I specific glomus cells revealed that a depletion of catecholamines and serotonin took place: PCPA lowered mainly serotonin, whereas reserpine depleted catechol- and indolamines about equally.It was shown by splitting of a few combined spectra with an iteration procedure of nonlinear regression, that serotonin affected slightly the height of the catecholamine peak. Thus, measuring the peak height in the catecholamine region of the total spectrum, the reduction of catecholamines to 64±22% by PCPA and to 13±4% by reserpine was statistically significant.These data are discussed in relation to possible mechanisms of action of the test substances and related to the content and nature of the biogenic amines in the carotid body.

Für technische Hilfe danken wir Fräulein B. Remki und Frau K. Wilp.     

Polyamine permeation and rectification of Kir4.1 channels

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.     

Detecting antigens by quantitative immuno-PCR

The quantitative immuno-PCR (qIPCR) technology combines the advantages of flexible and robust immunoassays with the exponential signal amplification power of PCR. The qIPCR allows one to detect antigens using specific antibodies labeled with double-stranded DNA. The label is used for signal generation by quantitative PCR. Because of the efficiency of nucleic acid amplification, qIPCR typically leads to a 10- to 1,000-fold increase in sensitivity compared to an analogous enzyme-amplified immunoassay. A standard protocol of a qIPCR assay to detect human interleukin 6 (IL-6) using a sandwich immunoassay combined with real-time PCR readout is described here. The protocol includes initial immobilization of the antigen, and coupling of this antigen with antibody-DNA conjugates is then carried out by (a) the stepwise assembly of biotinylated antibody, streptavidin and biotinylated DNA, (b) the use of a biotinylated antibody and an anti-biotin-DNA conjugate or (c) the employment of an anti-IL-6 antibody-DNA conjugate. Following the assembly of signal-generating immunocomplexes, real-time PCR is used to amplify and record the signal. Depending on the coupling strategy, the qIPCR assays require 4-7 h with only about 3 h hands-on-time. The use of qIPCR assays enables the detection of rare biomarkers in complex biological samples that are poorly accessible by conventional immunoassays. Therefore, qIPCR offers novel opportunities for the biomedical analysis of, for instance, neurodegenerative diseases and viral infections as well as new tools for the development of novel pharmaceuticals.     

Geometry strongly influences the response of numerical models of the lumbar spine--a probabilistic finite element analysis

Typical FE models of the human lumbar spine consider a single, fixed geometry. Such models cannot account for potential effects of the natural variability of the spine's geometry. In this study, we performed a probabilistic uncertainty and sensitivity analysis of a fully parameterized, geometrically simplified model of the L3-L4 segment. We examined the impact of the uncertainty in all 40 geometry parameters, estimated lower and upper bounds for the required sample size and determined the most important geometry parameters. The natural variability of the spine's geometry indeed strongly affects intradiscal pressure, range of motion and facet joint contact forces. Deriving generalized statements from fixed-geometry models as well as transferring those results to different cases thus can easily lead to wrong conclusions and should only be performed with extreme caution. We recommend a sample size of ≈ 100 to obtain reasonable accurate point estimates and a sufficient overview of the remaining uncertainties. Yet, only few parameters, especially those determining the disc geometry (disc height, end-plate width and depth) and the facets' position (intra-articular space, pedicle length, facet angles), proved to be truly important. Accurate measurement and modeling of those structures should therefore be prioritized.     

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 μm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.