Separation technique for the determination of 99Tc in milk and dairy products in case of emergency.

A method for the determination of (99)Tc (T1/2: 2.1 × 10(5)a) in milk and dairy products is presented in detail. The method includes the separation of fat and proteins from milk, the separation of Tc by anion exchange from other metals and the purification of Tc by extraction chromatography with subsequent measurement using LSC. A recommendation is given on how to use rhenium as a carrier for this particular matrix. The full analysis is done in 24h. The detection limit for milk is 0.2 Bq/l.

Light-switchable hemithioindigo-hemistilbene-containing peptides: ultrafast spectroscopy of the Z → E isomerization of the chromophore and the structural dynamics of the peptide moiety.

Two hemithioindigo-hemistilbene (HTI) derivatives, designed to operate as structural switches in peptides, as well as two HTI peptides are characterized by ultrafast spectroscopy in the visible and the infrared. The two HTI switches follow the reaction scheme published for other HTI compounds with a picosecond excited state reaction (τ(1) ≈ 6 ps) and isomerization from Z to E with τ(2) = 13 and 51 ps. As compared to the isolated chromophores, the isomerization reaction is slowed down in the chromopeptides to τ(2) = 24 and 69 ps. For the smaller peptide containing 6 amino acids, the structural changes of the peptide moiety observed via the IR spectrum in the amide I band follow the isomerization of the molecular switch closely. In the larger cyclic chromopeptide, containing 20 amino acids and mimicking a ß-hairpin structure in the Z-form of the chromophore, the peptide moiety also changes its structure during isomerization of the chromophore. However, the IR spectrum at the end of the observation period of 3 ns deviates significantly from the stationary difference spectrum. These signatures indicate that strong additional structural changes, e.g., breaking of interchain hydrogen bonds, also occur on longer time scales.

Occurrence of natural radioactivity in public water supplies in Germany: (238)U, (234)U, (235)U, (228)RA, (226)RA, (222)RN, (210)PB, (210)PO and gross alpha activity concentrations.

The Federal Office for Radiation Protection performed a representative survey on the radiological quality of drinking water in Germany. The aim of this study was to determine regional variations of natural radionuclide concentrations and to estimate radiation exposures caused by drinking water consumption. The study includes analyses of the natural radionuclides (238)U, (234)U, (235)U, (228)Ra, (226)Ra, (222)Rn, (210)Pb, (210)Po and of gross alpha activity concentrations in drinking water from 564 public water supplies. This represents 3 % of all German water supplies providing about 37 Mio. inhabitants. Results on ranges, medians and distributions of radionuclide concentrations of drinking water as well as age-dependent ingestion and inhalation doses estimated for members of the public are presented. Generally, the dose due to uranium isotopes is negligibly low. Radiation exposures are predominantly caused by (222)Rn, (228)Ra, (210)Po and (210)Pb. The ingestion dose deduced for adults (>17 a) and infants (0-1 a) is dominated by (222)Rn and (228)Ra, respectively. A gross alpha activity analysis procedure using liquid scintillation counting has been tested. Measured gross alpha activities values were found to be well related to the summarised activities of (238)U, (234)U, (226)Ra and (210)Po.

226Ra and 228Ra determination in mineral waters--comparison of methods.

A comparison of different radiochemical separation procedures and measurement techniques used to determine the activity concentration of (226)Ra and (228)Ra in water is made with respect to accuracy, detection limits and turn-around time. Radium-226 activity concentration was determined by the radon emanation technique, alpha-particle and gamma-ray spectrometry. To determine the (228)Ra activity concentration, four different techniques were used: low-level liquid scintillation counting, low-level proportional counting, alpha-particle and low-level gamma-ray spectrometry.

EC comparison on the determination of 226Ra, 228Ra, 234U and 238U in water among European monitoring laboratories.

In anticipation of new European requirements for monitoring radioactivity concentration in drinking water, IRMM organized an interlaboratory comparison on the determination of low levels of activity concentrations (about 10-100 mBq L(-1)) of the naturally occurring radionuclides (226)Ra, (228)Ra, (234)U and (238)U in three commercially available mineral waters. Using two or three different methods with traceability to the International System of Reference (SIR), the reference values of the water samples were determined prior to the proficiency test within combined standard uncertainties of the order of 3%-10%. An overview of radiochemical separation and measurement methods used by the 45 participating laboratories are given. The results of the participants are evaluated versus the reference values. Several of the participants' results deviate by more than a factor of two from the reference values, in particular for the radium isotopes. Such erroneous analysis results may lead to a crucial omission of remedial actions on drinking water supplies or to economic loss by an unjustified action.

Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain.

BACKGROUND AND PURPOSE: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands. EXPERIMENTAL APPROACH: We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays. KEY RESULTS: Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did. CONCLUSIONS AND IMPLICATIONS: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain.

BACKGROUND AND PURPOSE: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different. EXPERIMENTAL APPROACH: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay. KEY RESULTS: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. CONCLUSIONS AND IMPLICATIONS: The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).

Occludin binds to the SH3-hinge-GuK unit of zonula occludens protein 1: potential mechanism of tight junction regulation.

The interaction between tight junction proteins occludin and zona occludens protein 1 (ZO-1) was clarified. The sequence cc1 within the hinge region of ZO-1, connecting its SH3 and GuK domains, was identified as a new association site for the occludin C-terminus, core binding area GLRSSKRNLRKSR (mouse ZO-1(606-618)). Occludin also bound to the sequence H2 within GuK, core area HKLRKNNH (ZO-1(759-766)). In occludin, the binding core was ELSRLDKELDDYREESEEY (mouse occludin(455-473)). Helicity of the sequences was suggested by circular dichroism. Because basic residues in ZO-1, acidic residues in occludin (underlined), coiled-coil helix-forming leucine heptad motifs (bold) in occludin and, probably, in cc1 were essential, we conclude that interactions were both helical and ionic. Moreover, the GuK domain bound other GuK molecules, suggesting oligomerization of ZO-1. Generally, the assumption is supported that the SH3-hinge-GuK region represents a functional and regulatory unit in ZO-1 forming a multiprotein tight junction complex with occludin.

[Role of ORL1 receptors in the regulation of cardiac resistance to arrhythmogenic effects ]. / O znachenii ORL1-retseptorov v reguliatsii ustoichivosti serdtsa k aritmogennym vozdeistviiam.

It has been found that intravenous administration of nociceptin (0.4 mg/kg) prevents development of aconitine-induced arrhythmias but has no effect on the incidence of occlusion, reperfusion, CaCl2-induced arrhythmias, and exacerbates epinephrine-evoked dysrhythmias. Pretreatment with hexamethonium, atropine, guanethidine and naloxone did not abolish the arrhythmic effect of nociceptin. Intracerebroventricular infusion of orphanin FQ was shown to increase cardiac tolerance of arrhythmogenic influence of aconitine, but this effect is completely abolished by hexamethonium administration. It has been suggested that stimulation of both central and peripheral ORL1 receptors increases cardiac resistance against arrhythmogenic effect of aconitine via different mechanisms.

Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation.

Human lumbar CSF patterns of Abeta peptides were analysed by urea-based beta-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Abeta-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Abeta1-37/38/39 was found in addition to Abeta1-40 and Abeta1-42. Remarkably, Abeta1-38 was present at a higher concentration than Abeta1-42, being the second prominent Abeta peptide species in CSF. Patients with Alzheimer's disease (AD, n = 12) and patients with chronic inflammatory CNS disease (CID, n = 10) were differentiated by unique CSF Abeta peptide patterns from patients with other neuropsychiatric diseases (OND, n = 37). This became evident only when we investigated the amount of Abeta peptides relative to their total Abeta peptide concentration (Abeta1-x%, fractional Abeta peptide pattern), which may reflect disease-specific gamma-secretase activities. Remarkably, patients with AD and CID shared elevated Abeta1-38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE epsilon4 alleles resulted in an overall reduction of CSF Abeta peptides, which was pronounced for Abeta1-42. The severity of dementia was significantly correlated to the fractional Abeta peptide pattern but not to the absolute Abeta peptide concentrations.

Elevation of beta-amyloid peptide 2-42 in sporadic and familial Alzheimer's disease and its generation in PS1 knockout cells.

Urea-based beta-amyloid (Abeta) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Abeta peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Abeta peptides, namely, 1-40/42 and carboxyl-terminal-truncated 1-37, 1-38, and 1-39, was found. Besides Abeta1-42, we also observed a consistent elevation of amino-terminal-truncated Abeta2-42 in a detergent-soluble pool in brains of subjects with Alzheimer's disease. Abeta2-42 was also specifically elevated in cerebrospinal fluid samples of Alzheimer's disease patients. To decipher the contribution of potential different gamma-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Abeta peptides, we overexpressed beta-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1-/- neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1-/- neurons and PS1+/+ neurons overexpressing APP-Deltact (a slow-internalizing mutant) show a decrease of all secreted Abeta peptide species, as expected, because this mutant is processed mainly by alpha-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Abeta2-42 is significantly less affected in PS1-/- neurons and in neurons transfected with the endocytosis-deficient APP-Deltact construct. Our data confirm that PS1 is closely involved in the production of Abeta1-40/42 and the carboxyl-terminal-truncated Abeta1-37, Abeta1-38, and Abeta1-39, but the amino-terminal-truncated and carboxyl-terminal-elongated Abeta2-42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Abeta peptide species could be generated by a beta(Asp/Ala)-secretase activity.

NMR structure of the "ball-and-chain" domain of KCNMB2, the beta 2-subunit of large conductance Ca2+- and voltage-activated potassium channels.

The auxiliary beta-subunit KCNMB2 (beta(2)) endows the non-inactivating large conductance Ca(2+)- and voltage-dependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BKbeta(2)N) using NMR spectroscopy and patch clamp recordings. BKbeta(2)N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK alpha-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BKbeta(2)N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20-45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BKbeta(2)N-mediated inactivation.

Application of different surface plasmon resonance biosensor chips to monitor the interaction of the CaM-binding site of nitric oxide synthase I and calmodulin.

Surface plasmon resonance biosensors depend on modified gold surfaces to allow immobilization of proteins or peptides for interaction analysis. We investigated sensor chip surfaces that differ in the geometry of the immobilization matrix: two contain a three-dimensional coupling matrix and two have a surface with immobilization sites on a two-dimensional plane. Properties of sensor chips were compared by studying the interaction of calmodulin with a peptide representing the calmodulin-binding site of nitric oxide synthase I. Apparent K(D) values were determined by three different procedures in order to apply tests for self-consistency. At low surface densities (5-8 fmol/mm(2)) on three of the four tested surfaces, estimated K(D) values were within one order of magnitude and similar to the value found in solution (K(D) = 1-3 nM). When immobilization densities were increased by one to two orders of magnitude, apparent association rate constants were less distorted on a flat carboxymethylated surface than on dextran-coated sensor chips.

Optimization of the antimicrobial activity of magainin peptides by modification of charge.

Investigation of magainin II amide analogs with cationic charges ranging between +3 and +7 showed that enhancement of the peptide charge up to a threshold value of +5 and conservation of appropriate hydrophobic properties optimized the antimicrobial activity and selectivity. High selectivity was the result of both enhanced antimicrobial and reduced hemolytic activity. Charge increase beyond +5 with retention of other structural motifs led to a dramatic increase of hemolytic activity and loss of antimicrobial selectivity. Selectivity could be restored by reduction of the hydrophobicity of the hydrophobic helix surface (H(hd)), a structural parameter not previously considered to modulate activity. Dye release experiments with lipid vesicles revealed that the potential of peptide charge to modulate membrane activity is limited: on highly negatively charged 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol bilayers, reinforcement of electrostatic interactions had an activity-reducing effect. On neutral 1-palmitoyl-2-oleoylphosphatidylcholine bilayers, the high activity was determined by H(hd). H(hd) values above a certain threshold led to effective permeabilization of all lipid systems and even compensated for the activity-reducing effect of charge increase on highly negatively charged membranes.

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues.

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.

Peptide leucine arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin of the Northern Leopard frog, Rana pipiens.

On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.

Interaction of a mitochondrial presequence with lipid membranes: role of helix formation for membrane binding and perturbation.

The binding of a peptide to a biological membrane is often accompanied by a transition from a random coil structure to an amphipathic alpha-helix. Recently, we have presented a new approach which allows the determination of the thermodynamic parameters of membrane-induced helix formation [Wieprecht et al. (1999) J. Mol Biol. 294, 785]. It involves a systematic variation of the helix content of a given peptide by double D-substitution and a correlation of the binding parameters with the helicity. Here we have used this method to study membrane-induced helix formation for the presequence of rat mitochondrial rhodanese (RHD). The thermodynamic parameters of binding of the peptide RHD and of four of its double D-isomers were determined for 30 nm (SUVs) and 100 nm (LUVs) unilamellar vesicles composed of phosphatidylcholine/phosphatidylglycerol (3:1) using circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry. The incremental changes of the thermodynamic parameters of helix formation were found to be very similar for SUVs and LUVs. Membrane-induced helix formation of RHD entailed a negative enthalpy of Delta H(helix) = -0.5 to -0.6 kcal/mol/residue and was opposed by an entropy of about Delta S(helix) = -1 to -1.4 cal/mol K/residue. The free energy of helix formation, Delta G(helix), was about -0.2 kcal/mol, and helix formation accounted for 50-60% of the total free energy of membrane binding. Dye-release experiments were used to assess the role of helix formation for the membrane perturbation potential of the peptides. While helix formation plays a major role for membrane binding, it appears to have little importance for inducing membrane leakiness.

Evidence for downregulation of the endothelin-B-receptor by the use of fluorescent endothelin-1 and a fusion protein consisting of the endothelin-B-receptor and the green fluorescent protein.

We generated fusion proteins consisting of the endothelin-B (ET(B))-receptor and the enhanced green fluorescent protein (EGFP) to visualize receptor internalization. In Madin Darby canine kidney (MDCK) clones expressing ET(B)/EGFP fusion proteins, single class high affinity binding sites for [125I]endothelin-1 (ET-1) were found (for two different clones apparent K(D) values were 31 +/- 15 pM and 30 +/- 7 pM). Pretreatment of membranes with GTPgammaS prior to saturation analysis did not alter these values. We also labelled ET-1 with cyanine-dyes (Cy3/ET-1, Cy5/ET-1). In displacement analyses with membranes of MDCK ET(B)/EGFP clones using [125I]ET-1, we found reduced affinity for Cy3/ET-1 and Cy5/ET-1 (about 5- to 10-fold, respectively), but normal efficacy when compared to unlabelled ET-1. Both fluorescent ligands and the ET(B)/EGFP fusion protein were suitable for analysis of receptor trafficking in living cells and cells fixed at different timepoints. Laser scanning microscopy of MDCK ET(B)/EGFP clones incubated with Cy3/ET-1 or Cy5/ET-1 revealed rapid internalization of ligand/receptor complexes, which clustered in large, perinuclear structures (most probably late endosomes). Our data argue against recycling of the ET(B) receptor and favour its targeting to the lysosomal pathway.

Late endosomal/lysosomal targeting and lack of recycling of the ligand-occupied endothelin B receptor.

A fusion protein consisting of the endothelin B (ET(B)) receptor and the enhanced green fluorescent protein (EGFP) in conjunction with Cyanin3- or fluorescein-conjugated endothelin 1 (Cy3-ET1, Fluo-ET1) was used to investigate the ligand-mediated internalization of the ET(B) receptor. The ET(B) receptor and the ET(B)/EGFP fusion protein displayed very similar pharmacological properties when expressed in Chinese hamster ovary cells. The integrity of the fusion protein was verified by low temperature PAGE analysis of the (125)I-ET1-bound ET(B) receptor and the (125)I-ET1-bound ET(B)/EGFP fusion protein. Fluorescence microscopy of Chinese hamster ovary cells expressing the ET(B)/EGFP fusion protein demonstrated strong signals at the plasma membrane. On addition of Cy3-ET1, internalization of ligand and receptor occurred within 5 min via a sucrose-sensitive (i.e., clathrin-mediated) pathway. On further incubation, ET(B)/EGFP and Cy3-ET1 fluorescences were found in the perinuclear region, colocalized with fluorescent low density lipoproteins, a marker of the late endosomal/lysosomal pathway, but not with fluorescent transferrin, a marker of the recycling pathway. No dissociation of Cy3-ET1 from the receptor was seen within 4 h. Using (125)I-ET1 or Cy3-ET1, binding sites were again demonstrable at the cell surface within 2 h. The reappearance of binding sites was abolished by prior treatment of the cells with cycloheximide, an inhibitor of protein synthesis. The data demonstrate that the ligand-occupied ET(B) receptor is internalized; however, it does not recycle like most of the G protein-coupled receptors but is sorted to the late endosomal/lysosomal pathway in a manner similar to that of the family of protease-activated receptors.