Development of a high efficiency proton source for the Frankfurter-Neutronen-Quelle am Stern-Gerlach-Zentrum.

A new version of high efficiency proton source is being developed for Frankfurter-Neutronen-Quelle am Stern-Gerlach-Zentrum, a worldwide unique pulsed neutron source. The injector will provide dc proton beam currents of 200 mA at 120 keV with a beam divergence angle of less than 50 mrad. The new design consists of a plasma generator with a multiple filament arrangement and a compact pentode extraction system. The beam will be extracted from a seven hole outlet electrode. Great efforts are made to achieve an adequate operation time (~3 days) of the source as well as to obtain high reliability during that operation period with less than 1 V breakdowns per hour. The following article will present the current status of the proton source development including beam formation simulations with IGUN.

Improvement of lay rescuer chest compressions with a novel audiovisual feedback device : A randomized trial.

BACKGROUND: Bystander actions and skills determine among others the outcome of out-of-hospital cardiac arrest. However, the depth and rate of chest compressions (CC) are difficult to estimate for laypeople and poor CC quality may result. Our study aimed to evaluate the impact of a new feedback device on CC performance by laypeople. The percentage of CC with both correct rate and correct depth of all CC served as primary endpoint. METHODS: Forty-eight subjects with no medical background performed 2 min of CC on a manikin with and without a novel feedback device (TrueCPR™, Physio-Control, Redmond, Wash.). The device uses a novel, non-accelerometer-based technology. Participants were randomized into two groups. Group 1 performed a 2-min CC trial first with audiovisual feedback followed by a trial with no feedback information, while group 2 performed the task in reverse order. RESULTS: The absolute percentage of CC with correct rate and depth was significantly higher with the use of the device (59 ± 34% vs. 15 ± 21%, p < 0.0001). The longest interval without correct CC was significantly decreased (76.5 s vs. 27.5 s, p < 0.0001). CONCLUSION: The quality of CC carried out by laypeople is significantly improved with the use of a new feedback device. The device may be useful for cardiopulmonary resuscitation (CPR) by laypeople and for educational purposes.

Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.

It is well established that the terminal renal collecting duct is capable of electrogenic Na+ absorption. The present experiments examined other active ion transport processes in primary cultures of the rat inner medullary collecting duct. When the amiloride analogue benzamil inhibited electrogenic Na+ absorption, cAMP agonists stimulated a transmonolayer short circuit current that was not dependent on the presence of Na+ in the apical solution, but was dependent on the presence of Cl- and HCO3-. This current was not inhibited by the loop diuretic bumetanide, but was inhibited by ouabain, an inhibitor of the Na+/K+ pump. The current was reduced by anion transport inhibitors, with a profile similar to that seen for inhibitors of the cystic fibrosis transmembrane conductance regulator (CFATR) Cl- channel. Using several PCR strategies, we demonstrated fragments of the predicted lengths and sequence identity with the rat CFTR. Using whole-cell patch-clamp analysis, we demonstrated a cAMP-stimulated Cl- current with characteristics of the CFTR. We conclude that the rat inner medullary collecting duct has the capacity to secrete anions. It is highly likely that the CFTR Cl- channel is involved in this process.

rENaC is the predominant Na+ channel in the apical membrane of the rat renal inner medullary collecting duct.

The terminal nephron segment, the inner medullary collecting duct (IMCD), absorbs Na+ by an electrogenic process that involves the entry through an apical (luminal) membrane Na+ channel. To understand the nature of this Na+ channel, we employed the patch clamp technique on the apical membrane of primary cultures of rat IMCD cells grown on permeable supports. We found that all ion channels detected in the cell-attached configuration were highly selective for Na+ (Li+) over K+. The open/closed transitions showed slow kinetics, had a slope conductance of 6-11 pS, and were sensitive to amiloride and benzamil. Nonselective cation channels with a higher conductance (25-30 pS), known to be present in IMCD cells, were not detected in the cell-attached configuration, but were readily detected in excised patches. The highly selective channels had properties similar to the recently described rat epithelial Na+ channel complex, rENaC. We therefore asked whether rENaC mRNA was present in the IMCD. We detected mRNA for all three rENaC subunits in rat renal papilla and also in primary cultures of the IMCD. Either glucocorticoid hormone or mineralocorticoid hormone increased the amount of alpha-rENaC subunit mRNA but had no effect on the mRNA level of the beta-rENaC or gamma-rENaC subunits. From these data, taken in the context of other studies on the characteristics of Na+ selective channels and the distribution of rENaC mRNA, we conclude that steroid stimulated Na+ absorption by the IMCD is mediated primarily by Na+ channels having properties of the rENaC subunit complex.

CD95 maintains stem cell-like and non-classical EMT programs in primary human glioblastoma cells.

Glioblastoma (GBM) is one of the most aggressive types of cancer with limited therapeutic options and unfavorable prognosis. Stemness and non-classical epithelial-to-mesenchymal transition (ncEMT) features underlie the switch from normal to neoplastic states as well as resistance of tumor clones to current therapies. Therefore, identification of ligand/receptor systems maintaining this privileged state is needed to devise efficient cancer therapies. In this study, we show that the expression of CD95 associates with stemness and EMT features in GBM tumors and cells and serves as a prognostic biomarker. CD95 expression increases in tumors and with tumor relapse as compared with non-tumor tissue. Recruitment of the activating PI3K subunit, p85, to CD95 death domain is required for maintenance of EMT-related transcripts. A combination of the current GBM therapy, temozolomide, with a CD95 inhibitor dramatically abrogates tumor sphere formation. This study molecularly dissects the role of CD95 in GBM cells and contributes the rational for CD95 inhibition as a GBM therapy.

Perinatal morbidity in women with undiagnosed gestational diabetes in northern thuringia in Germany.

INTRODUCTION: Undiagnosed gestational diabetes mellitus (GDM) is associated with severe perinatal complications. PATIENTS AND METHODS: Out of 970 women, infant and maternal morbidity was assessed in 114 mother-children-pairs with an infant birth weight over the 90th percentile (Voigt et al., 1996). It was the aim of this retrospective study to assess the number of mothers with undiagnosed GDM, who have born a macrosomic child. RESULTS: The macrosomia rate in newborns was 12 % in this study excluding macrosomic infants of mothers with preexisting diabetes mellitus. Maternal data: Age 28.3 +/- 5.3 years, adipositas (body-mass-index > 30 kg/m) in 42.1 % vs. 30.4 % in the peer group (p < 0.02), increase in weight > 15 kg during pregnancy in 57.9 % of the mothers who have given birth to a macrosomic child vs. 30.9 % in the peer group (p < 0.0001), family history of diabetes mellitus (28.0 % vs. 11.3 % in the peer group, p = 0.006), preeclampsia in 8.8 % vs. 2.7 % in the peer group (p = 0.002), cervical insufficiency in 2.6 % vs. 0.4 % in the peer group (p = 0.02). After delivery HbA1c was elevated in 38.6 % of the women having born macrosomic infant (mean HbA1c: 5.0 % +/- 0.5). Infant data: neonatal jaundice 16.7 % vs. 4.5 % in the peer group, p < 0.0001. There were no statistically significant differences concerning perinatal condition and malformations. Neonatal hypoglycaemia occurred in 9.6 % of the macrosomic infants. Cord blood insulin levels were significantly elevated in comparison to the peer group of mothers without metabolic disorders and having born eutroph infants (8.4 mU/l [3.0 - 100.0] vs. 5.3 mU/l [3.0 - 30.7], p = 0.01). 11.4 % of all macrosomic infants had cord blood insulin levels above the normal range. CONCLUSION: More than one third of the mothers having born one or more macrosomic infants had an impairment of glucose metabolism immediately after birth. The elevated prevalence of preeclampsia in this group confirms the relationship of hypertension and impaired glucose metabolism during pregnancy. The detection of hyperinsulinaemia, postnatal hypoglycaemia, elevated prevalence of neonatal jaundice with need of further therapy and diabetic fetopathy in macrosomic infants of mothers, whose metabolism was not monitored during pregnancy, pinpoint the need for a diagnostic screening for GDM.

Evidence that the FSH receptor itself is not a calcium channel.

FSH has been shown to stimulate the uptake of calcium in cultured rat Sertoli cells, resulting in an increase in cytosolic calcium concentrations. One possibility which has been put forth is that the FSH receptor per se may act as a calcium channel. This is all the more tantalizing given the proposed structure of this receptor which, like all other G protein-coupled receptors, is thought to have the putative transmembrane helices forming a bundle-like structure in the plasma membrane. To test whether the FSH receptor could function as a calcium channel, we performed whole-cell voltage clamp experiments on 293 and 293F(wt1) cells, which are a clonal line of 293 cells expressing high levels of rat FSH receptors. The 293 cells, which do not express FSH receptors, were found to lack any detectable inward calcium currents, and therefore, serve as an excellent model for transfecting with potential calcium conducting FSH receptors. When the 293F(wt1) cells were then tested, no inward calcium currents could be detected in either control or FSH-stimulated cells. These results suggest that the FSH receptor itself is not a calcium channel and, therefore, FSH must be stimulating endogenous calcium channels in rat Sertoli cell plasma membranes.

Inhibitors of cholesterol biosynthesis. 2. Hypocholesterolemic and antioxidant activities of benzopyran and tetrahydronaphthalene analogues of the tocotrienols.

Tocotrienols exhibit antioxidant and cholesterol-biosynthesis-inhibitory activities and may be of value as antiatherosclerotic agents. The mechanism of their hypolipidemic action involves posttranscriptional suppression of HMG-CoA reductase (HMGR) in a manner mimicking the action of putative non-sterol feedback inhibitors. The in vitro cholesterol-biosynthesis-inhibitory and HMGR-suppressive activities in HepG2 cells of an expanded series of benzopyran and tetrahydronaphthalene isosteres and the hypocholesterolemic activity of selected compounds assessed in orally dosed chickens are presented. Preliminary antioxidant data of these compounds have been obtained using cyclic voltammetry and Cu-induced LDL oxidation assays. The farnesyl side chain and the methyl/hydroxy substitution pattern of gamma-tocotrienol deliver a high level of HMGR suppression, unsurpassed by synthetic analogues of the present study. In orally dosed chickens, 8-bromotocotrienol (4o), 2-desmethyltocotrienol (4t), and the tetrahydronaphthalene derivative 35 exhibit a greater degree of LDL cholesterol lowering than the natural tocotrienols.

Investigations of the cholinergic modulation of GABA release in rat thalamus slices.

The thalamus receives a dense cholinergic projection from the pedunculopontine tegmentum. A number of physiological studies have demonstrated that this projection causes a dramatic change in thalamic activity during the transition from sleep to wakefulness. Previous anatomical investigations have found that muscarinic type 2 receptors are densely distributed on the dendritic terminals of GABAergic interneurons, as well as the somata and proximal dendrites of GABAergic cells in the thalamic reticular nucleus. Since these structures are the synaptic targets of cholinergic terminals in the thalamus, it appears likely that thalamic pedunculopontine tegmentum terminals can activate muscarinic type 2 receptors on GABAergic cells. To test whether activation of muscarinic type 2 receptors affects the release of GABA in the thalamus, we have begun pharmacological studies using slices prepared from the rat thalamus. We have found that the application of the nonspecific muscarinic agonist, methacholine, and the muscarinic type 2-selective agonist, oxotremorine.sesquifumarate, diminished both the baseline, and K(+) triggered release of [(3)H]GABA from thalamic slices. This effect was calcium dependent, and blocked by the nonselective muscarinic antagonist atropine, the muscarinic type 2-selective antagonist, methoctramine, but not the muscarinic type 1 antagonist, pirenzepine. Thus, it appears that one function of the pedunculopontine tegmentum projection is to decrease the release of GABA through activation of muscarinic type 2 receptors. This decrease in inhibition may play an important role in regulating thalamic activity during changes in states of arousal.

SGK is a primary glucocorticoid-induced gene in the human.

Serum- and glucocorticoid-induced kinase (sgk) is transcriptionally regulated by corticosteroids in several cell types. Recent findings suggest that sgk is an important gene in the early action of corticosteroids on epithelial sodium reabsorption. Surprisingly, the human sgk was reported not to be transcriptionally regulated by corticosteroids in a hepatoma cell line, and thus far no glucocorticoid response element has been identified in the human SGK gene. Since humans clearly respond to both aldosterone and glucocorticoids in cells where sgk action seems to be important, in this study we determined sgk mRNA levels following dexamethasone treatment for various duration in five human cell lines. These cell lines included epithelial cells (H441, T84 and HT29) and lymphoid/monocyte (U937 and THP-1) lines. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that sgk mRNA levels are markedly induced by glucocorticoids in all of the five cell lines studied. Time course analyses revealed that sgk mRNA levels are elevated as early as 30 min after addition of the glucocorticoid, and remain elevated for several hours. Northern analysis in H441 cells confirmed that sgk is an early induced gene. The induction of sgk by dexamethasone was unaffected by cycloheximide, indicating that it does not require de novo protein synthesis. These results indicate that the human sgk, just like its counterparts in other species, is a primary glucocorticoid-induced gene.

Analysis of monoclonal antibody and immunoconjugate digests by capillary electrophoresis and capillary liquid chromatography.

Comparative peptide mapping of a monoclonal antibody chimeric BR96 and corresponding doxorubicin (DOX) immunoconjugate was performed using capillary electrophoresis (CE) and capillary liquid chromatography (CLC). A unique, highly sensitive and selective approach combined with both UV absorbance and laser-induced fluorescence (LIF) detection has been developed and applied to studies including enzymatic digests of antibody and conjugate and related drug and conjugation linker substances. The analytical methodology has been established based on the unique characteristic of the anticancer drug DOX which yields native fluorescence. With an excitation wavelength of 488 nm from argon-ion laser, DOX conjugated to the monoclonal antibody using a hydrazone linker can be determined with a detection limit at the attomole level. Approaches were developed based on the successful conjugation and analysis of a model peptide conjugate. Enzymatic digests of the monoclonal antibody BR96 and its immunoconjugate were mapped by CE and CLC with on-line UV and LIF detection, which results in a unique fingerprint for structural analysis. With a two-dimensional LC-CE approach, conjugated peptide-DOX species from LC were further analyzed by CE with LIF detection. The drug-containing peptide fragments in the mixture were readily detected, which can be further characterized using other complementary analytical techniques.

Affinity capillary electrophoresis applied to the studies of interactions of a member of heat shock protein family with an immunosuppressant.

The bioaffinity of receptor-ligand interactions is investigated by determining the binding constant (association constant or dissociation constant) of the resulting complex utilizing affinity capillary electrophoresis (ACE). The ACE binding assay was established with a potent immunosuppressant, deoxyspergualin (DSG), that binds specifically to Hsc70, a constitutive or cognate member of heat shock protein 70 (Hsp70) family. Quantitative determination of binding constants under different running buffer systems provide comparative results. The association constants for the interaction between Hsc70 protein and DSG were found to be 5.7 x 10(4) M-1 in a buffer with pH 6.95 and 6.3 x 10(4) M-1 in a buffer with pH 5.30 (or corresponding dissociation constants, 18 and 16 microM, respectively) based on Scatchard analyses. Binding of DSG to a synthetic peptide, SINPDEAVAYGAAVQAAILSGDK, one of the DSG-binding fragments found from tryptic digest of Hsc70 protein, provides further detailed information for the understanding of Hsc70 binding domain. The applicability of using coated capillaries was also evaluated for probing Hsc70-DSG interaction.

Manumycins E, F and G, new members of manumycin class antibiotics, from Streptomyces sp.

Three new manumycin class antibiotics, namely manumycins E, F and G, were isolated from the culture broth of Streptomyces sp. strain WB-8376. Their structures were established by spectroscopic methods, and the S configuration of C-4 in the epoxycyclohexenone moiety was determined by CD exciton chirality method for each of the three compounds. Manumycins E, F and G are active against Gram-positive bacteria, and have moderate inhibitory effects on the farnesylation of p21 ras protein. They demonstrated weak cytotoxic activity against human colon tumor cell HCT-116.

On-line mass spectrometric investigation of the peroxidase-catalysed oxidation of uric acid.

The enzymatic and electrochemical oxidation pathways of uric acid were determined on-line with thermospray-tandem mass spectrometry. Products and intermediates formed as a result of electrooxidation were monitored as the electrode potential was varied. Electrochemical results served as a model for the enzymatic studies. In fact, electrochemical studies were essential for elucidating the structures of intermediates because of the high conversion efficiencies in electrooxidation. Products and intermediates formed as a result of enzymatic oxidation of uric acid were monitored as the reaction time was varied. When the enzymatic oxidation of uric acid with peroxidase and H2O2 was studied, the same intermediates and products were observed as in the electrochemical oxidation. The tandem mass spectrometric results provide convincing evidence that the primary intermediate produced during both the enzymatic and electrochemical oxidation of uric acid has a quinonoid diimine structure. The primary intermediate can follow three distinct reaction pathways to produce the identified final products. The final enzymatic and electrochemical oxidation products observed in these studies were urea, CO2, alloxan, alloxan monohydrate, allantoin, 5-hydroxyhydantoin-5-carboxamide and parabanic acid.

Miniaturized HPLC and ionspray mass spectrometry applied to the analysis of Paclitaxel and taxanes.

Analysis of the antitumor agent Paclitaxel, related taxane analogues and yew tree bark extracts has been carried out using an HPLC system capable of performing chromatographic separations with conventional, small-bore, and micro-bore columns. Both diode array detector and mass spectrometry were incorporated into this system, providing additional spectral and structural information for identification of unknown samples. In conjunction with some basic theoretical studies dealing with miniaturized HPLC systems, experiments were designed to minimize the contribution of extra-column variances. Three chromatographic columns, 4.6, 2 and 1 mm i.d., were elevated using a standard mixture consisting of Paclitaxel and three analogues. The experimental results obtained in these columns demonstrated good correlation with theoretical calculations with respect to the sensitivity enhancement. Studies on the combination of miniaturized HPLC with ionspray mass spectrometry for Paclitaxel samples showed dramatic improvement of MS performance as compared to conventional LC/MS. The advantages of this miniaturized LC/MS system are evidenced by enhanced mass sensitivity, which was more that two order of magnitude higher when changed from a 4.6 mm i.d. column to a 2.0 mm i.d. column, greatly improved peak shape, and the potential gain of efficiency. These studies demonstrate great potential of miniaturized HPLC/MS systems for structural characterization and confirmation of various pharmaceutical compounds.

Monitoring in vitro experiments using microdialysis sampling on-line with mass spectrometry.

A method has been developed for the real-time analysis of components in in vitro reactions by the on-line combination of microdialysis sampling (MD) with tandem mass spectrometry (MS/MS) and single stage mass spectrometry (MS). Apparatus and parameters associated with the integration have been studied. Analytical figures of merit for the drug gepirone have been determined. The qualitative 'limit of identification' was found to be 100 ng/ml and 200 ng/ml for methods using thermospray and electrospray MS interfaces, respectively. Using this approach, monitoring of in vitro experiments involving drug metabolites, enzymatic reactions, and ligand-protein binding interactions were performed.

Profiling impurities and degradants of butorphanol tartrate using liquid chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry substructural techniques.

A rapid and systemic strategy based on liquid chromatography/mass spectrometry (LC/MS) profiling and liquid chromatography/tandem mass spectrometry (LC/MS/MS) substructural techniques was utilized to elucidate the degradation products of butorphanol, the active ingredient in stadol NS. This strategy integrates, in a single instrumental approach, analytical HPLC, UV detection, full-scan electrospray mass spectrometry, and tandem mass spectrometry to rapidly and accurately elucidate structures of impurities and degradants. In these studies, several low-level degradation products were observed in long-term storage stability samples of bulk butorphanol. The resulting analytical profile includes information on five degradants including molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. The degradation products formed during long-term storage of butorphanol tartrate included oxidative products proposed as 9-hydroxy-and 9-keto-butorphanol, norbutorphanol, a ring-contraction degradant, and delta 1, 10 a-butorphanol. These methodologies are applicable at any stage of the drug product cycle from discovery through to development. This library of butorphanol degradants provides a foundation for future development work regarding product monitoring, as well as a useful diagnostic tool for new degradation products.

Analysis of amino acid enantiomers derived from antitumor antibiotics using chiral capillary electrophoresis.

The chiral separation of enantiomeric forms of derivatized amino acids have been achieved based on a metalchelate chiral capillary electrophoretic method and a cyclodextrin mediated host-guest interaction approach in micellar electrokinetic chromatography (MEKC) mode with laser-induced fluorescence detection. This approach has been applied to the determination of enantiomeric forms of amino acids derived from novel depsipeptide antitumor antibiotics, BMY-45012 and its analogs. Amino acids were analyzed by complete hydrolysis and the hydrolysate was derivatized with either dansyl chloride for UV absorbance detection or fluorescein isothiocyanate for laser based fluorescence detection. The presence of several amino acids, serine and beta-hydroxyl-N-methy-valine in the proposed structure have been confirmed as D-serine and L-beta-hydroxyl-N-methy-valine enantiomeric forms by both chiral capillary electrophoresis (chiral CE) and MEKC approaches. A non-chiral amino acid, sarcosine, was also confirmed. These methodologies provide a quick and sensitive approach for the determination of amino acids racemization of pharmaceutical natural products and have proven to be useful for structural elucidation refinement.

Predictive strategy for the rapid structure elucidation of drug degradants.

Structural information on drug degradants and impurities can serve to accelerate the drug discovery and development cycle. Traditional structure elucidation methodologies for obtaining this information are often slow and resource-consuming; therefore, LC/MS profiling and LC/MS/MS substructural analysis methodologies have been developed to rapidly and accurately elucidate structures of impurities and degradants. This work is a further development of methodologies used for the elucidation of degradation products of paclitaxel [K.J. Volk et al., Proc. 9th AAPS Ann. Meeting, 1994, p.29]. In this study cefadroxil was used as a model compound for the evaluation of a predictive strategy for the production and elucidation of impurities and degradants induced by acid, base, and heat, using LC/MS and LC/MS/MS profiling methodology, resulting in an LC/MS degradant database which includes information on molecular structures, chromatographic behavior, molecular weight, UV data, and MS/MS substructural information. Furthermore, libraries such as this can provide a predictive foundation for pre-clinical development work involving drug stability, synthesis, and monitoring.

A high intensity 200 mA proton source for the FRANZ-Project (Frankfurt-Neutron-Source at the Stern-Gerlach-Center).

At the University of Frankfurt a high current proton source has been developed and tested for the FRANZ-Project [U. Ratzinger, L. P. Chau, O. Meusel, A. Schempp, K. Volk, M. Heil, F. Käppeler, and R. Stieglitz, "Intense pulsed neutron source FRANZ in the 1-500 keV range," ICANS-XVIII Proceedings, Dongguan, April 2007, p. 210]. The ion source is a filament driven arc discharge ion source. The new design consists of a plasma generator, equipped with a filter magnet to produce nearly pure proton beams (92 %), and a compact triode extraction system. The beam current density has been enhanced up to 521 mA/cm(2). Using an emission opening radius of 4 mm, a proton beam current of 240 mA at 50 keV beam energy in continuous wave mode (cw) has been extracted. This paper will present the current status of the proton source including experimental results of detailed investigations of the beam composition in dependence of different plasma parameters. Both, cw and pulsed mode were studied. Furthermore, the performance of the ion source was studied with deuterium as working gas.