Factor VIII Bypassing Activity (FEIBA) assays: standardization and development of the 1st NIBSC Working Standard for FEIBA--results from a collaborative study.

Factor-Eight-Inhibitor-Bypassing-Activity (FEIBA) is a bypassing-agent used to control spontaneous bleeding or cover surgical interventions in Haemophiliacs who develop neutralizing antibodies against FVIII/FIX. The market lot-release of FEIBA is dependent on specific clot-based assays, carried out by both the manufacturer and regulatory authorities, relative to manufacturer's in-house standards, which are produced on a small-scale and are replaced frequently. We sought to standardize the FEIBA assay by developing a FEIBA primary standard which would be internationally available in sufficiently large quantities, with a predicted lifetime of many years. A collaborative study involving the manufacturer and three regulatory authorities, was carried out in which a candidate material, sample B (06/172), was calibrated by assays relative to the manufacturer's in-house FEIBA standards (C and D). All laboratories used their routine validated methods (16 APTT-assays, 8 ACTIN-FS-assays and 27 DAPTTIN-assays). Intra-laboratory geometric coefficients of variation (GCVs) for candidate B ranged from 3% to 29% (GCVs <9% from majority of labs). Assessment of inter-laboratory variability gave overall GCV values of 6.9% and 4.4% relative to standards C and D, respectively, for all methods. There was good agreement in potency estimation between laboratories using each of the three methods, with the overall potencies by the three methods differing by less than 10% of the overall mean, giving an overall combined potency of 28.0 units per ampoule. All participants agreed that candidate B (06/172) be established as the 1st NIBSC Working Standard for FEIBA with an assigned potency of 28.0 units per ampoule, based on combined results for both methods, relative to either standard C or D.

Differential pulse polarographic investigation of the antifungal drugs itraconazole, ketoconazole, fluconazole and voriconazole using a dropping mercury electrode.

The electrochemical reactions of the antifungal drugs itraconazole, ketoconazole, fluconazole and voriconazole have been investigated by differential pulse polarography (DPP) using a dropping mercury electrode (DME). All investigations were carried out in Britton-Robinson buffer solutions and methanol with varying pH values. Ketoconazole and itraconazole both showed a reduction peak with a potential between -1.5V and -1.6 V. Stable and reproducible conditions for the determination of itraconazole (c = 1 x 10(-7) M) were found within the pH range of 6.0 to 8.0 and for the determination of ketoconazole (c = 5 x 10(-8) M) within pH 6.0 to 7.0. Voriconazole showed a reduction peak with a peak potential of -1.7 V (c = 1 x 10(-5) M) within the pH range of 8.0 to 10.0. In the case of fluconazole no electrochemical activity was found.

Emission properties and photon statistics of a single quantum dot laser.

A theoretical description for a single quantum-dot emitter in a microcavity is developed.We analyze for increasing steady-state pump rate the transition from the strong-coupling regime with photon antibunching to the weak-coupling regime with coherent emission. It is demonstrated how Coulomb interaction of excited carriers and excitation-induced dephasing can strongly modify the emission properties. Our theoretical investigations are based on a direct solution of the Liouville-von Neumann equation for the coupled carrier-photon system. We include multiple carrier excitations in the quantum dot, their Coulomb interaction, as well as excitation-induced dephasing and screening. Similarities and differences to atomic systems are discussed and results in the regime of recent experiments are interpreted.

Differential pulse polarographic investigation of lansoprazole and rabeprazole using dropping mercury electrode.

The electrochemical reduction of the proton pump inhibitors (PPI) lansoprazole and rabeprazole has been investigated by differential pulse polarography (DPP) using a dropping mercury electrode (DME). The results were compared not only among both substances but also among other proton pump inhibitors depending on the varying chemical structures of the agents. All investigations were carried out in Britton-Robinson buffer solutions with pH values from 3.0 to 11.0. It was shown that both PPI undergo an extensive decomposition decreasing with increasing pH values forming two main compounds, a cyclic sulfenamide and a dimer. In this case lansoprazole was found to be stable at pH 8.0 and rabeprazole at pH 9.0. The decomposition of rabeprazole ran considerably quicker and also up to higher pH values than those of lansoprazole. The peak currents varied linearly with the concentration of both PPI in the range from 1 x 10(-6) M to 7 x 10(-5) M at pH 9.0. Both substances showed similarities in reaction as well as individual differences based on their varying chemical structures and characteristics.

Regular molecular nano-dot patterns for RW-data storage in the optical near field.

In this contribution further steps to realize our concept of optical data storage in molecular nano-dots as storage bits are reported. A preparation method has been developed successfully to achieve regular nano-dot patterns of high density. This is considered a breakthrough because regularity is prerequisite for the use of nano-dots in storage media. Optical imaging of dot patterns and individual writing/reading access is realized by confocal and near-field optical microscopy (fluorescence detection mode). Examples for regular patterns at different dot densities are shown. Perspectives towards terabit per square inch density are outlined.

Real-time Bose-Einstein condensation in a finite volume with a discrete spectrum.

We show that Bose condensation in real time occurs in a finite system not only as an accumulation of the bosons in the ground state below a critical temperature, but also as a rapid enhancement of an arbitrary small symmetry breaking, followed by a very slow decay of the symmetry breaking order parameter from the almost ideal value to the vanishing equilibrium value. We show this analytically on an exactly soluble model and numerically on a model of noninteracting bosons in an oscillator potential.

Identification of various urinary metabolites of fluorene using derivatization solid-phase microextraction.

A method for the qualitative analysis of various metabolites of fluorene is presented. The method uses solid-phase microextraction with an 85 microm polyacrylate fiber for extraction, headspace silylation with BSTFA and MTBSTFA without any catalyst for on-fiber derivatization and GC-MS in the selected ion monitoring mode for separation and detection. The suitability of the method for profile analysis of polycyclic aromatic hydrocarbon metabolites is shown by analyzing an urine of an occupationally exposed person after enzymatic cleavage of the excreted conjugates. Satisfactory separation of all investigated metabolites was achieved without interferences due to matrix peaks.

Subthreshold carrier-LO phonon dynamics in semiconductors with intermediate polaron coupling: a purely quantum kinetic relaxation channel.

Femtosecond transmission spectra of highly polar CdTe are compared to more covalent GaAs contrasting semiclassical kinetics with two-time quantum kinetics based on the Dyson equation. Nonequilibrium heavy holes in CdTe show ultrafast energy redistribution via the Fröhlich mechanism even if photoexcited below the LO phonon energy. This subthreshold relaxation is a genuine quantum kinetic effect. It gains importance if the polaron self-energy is comparable to the phonon energy. Conservation of the free-particle energies is not required under these conditions.

Bose-Einstein condensation quantum kinetics for a gas of interacting excitons.

A quantum kinetics of the Bose-Einstein condensation in the self-consistent (s.c.) Hartree-Fock-Bogoliubov (HFB) model of the interacting Bose gas is formulated and numerically solved for the example of excitons scattering with a thermal bath of acoustic phonons. The theory describes the condensation in real time starting from a nonequilibrium initial state towards the equilibrium HFB solution. The s.c. changes of the spectrum are automatically incorporated in the scattering terms.

Total synthesis of brevetoxin A.

Brevetoxin A is the most potent neurotoxin secreted by Gymnodinium breve Davis, a marine organism often associated with harmful algal blooms known as 'red tides'. The compound, whose mechanism of action involves binding to and opening of sodium channels, is sufficiently toxic to kill fish at concentrations of nanograms per ml and, after accumulation in filter-feeding shellfish, to poison human consumers. The precise pathway by which nature constructs brevetoxin A is at present unknown, but strategies for its total synthesis have been contemplated for some time. The synthetic challenge posed by brevetoxin A reflects the high complexity of its molecular structure: 10 oxygen atoms and a chain of 44 carbon atoms are woven into a polycyclic macromolecule that includes 10 rings (containing between 5 and 9 atoms) and 22 stereogenic centres. Particularly challenging are the 7-, 8- and 9-membered rings which allow the molecule to undergo slow conformational changes and force a 90 degrees twist at one of its rings. Here we describe the successful incorporation of methods that were specifically developed for the construction of these rings into an overall strategy for the total synthesis of brevetoxin A in its naturally occurring form. The convergent synthesis reported here renders this scarce neurotoxin synthetically available and, more importantly, allows the design and synthesis of analogues for further biochemical studies.

Inherent glucocorticoid response potential of isolated hypothalamic neuroendocrine neurons.

Within the broader framework of facilitating investigations into the inherent responses of restricted neuronal phenotypes devoid of their in vivo afferents, serum- and steroid-free cultures enriched in corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), and beta-endorphin (beta-END) peptidergic neurons were prepared from the hypothalamic paraventricular (PVN: CRH and AVP) and/or arcuate (ARC: beta-END) nuclei of juvenile male rats. The functional viability of these ARC/PVN cultures was verified by their ability to synthesize and secrete CRH, AVP, and beta-END under basal and depolarizing (veratridine) conditions in vitro. Peptide secretion was shown to be Ca2+ and Na+ dependent in that it was blocked in the presence of verapamil and tetrodotoxin, respectively. Exposure of ARC/PVN cocultures to the glucocorticoid dexamethasone (DEX) resulted in a dose-dependent increase of CRH secretion and an inhibition of AVP and beta-END; the CRH responses deviated strikingly from predictions based on in vivo experiments. Steroid withdrawal or treatment with the glucocorticoid receptor antagonist RU38486 reversed these trends. Opposite effects of DEX on CRH secretion were observed in cultures consisting of PVN cells only. Supported by studies using an opioid receptor agonist (morphine) and antagonist (naloxone), these observations demonstrate that ARC-derived (beta-END) neurons modulate the responses of PVN neurons to DEX.

La colectivización de la medicina y los medios de comunicación / The collectivization of medicine and the media

En esta ponencia el expositor se refiere en primer lugar a la importantísima labor que dichos medios cumplen en la sociedad y a los problemas que pueden acarrearse cuando esa labor se ejerce por fuera de las normas de objetividad y de ética que deben enmarcar el ejercicio de la profesión por parte del periodista. Se hace enseguida un recuento somero de la génesis de la Seguridad Social en el mundo, con énfasis en el origen del concepto moderno en Alemania, como consecuencia de la Revolución Industrial y la consiguiente aparición del proletariado urbano. Luego el ponente esboza el desarrollo de la Seguridad Social en Colombia a partir de 1946, centrándose en la organización del sistema de atención de la salud adoptado por el Instituto Colombiano de Seguros Sociales. Expone las que, en su concepto, constituyen características que afectaron profundamente el ejercicio de la medicina, al distorsionarse las bases esenciales del Acto Médico, como son la Libertad de elección y la confidencialidad. Analiza luego el deterioro que ha sufrido la imagen del médico ante la sociedad, debido al despliegue informativo parcializado o deformado de sucesos relacionados con la práctica médica. Finalmente, se refiere a las perspectivas que abre la Ley 100 de 1993, la cual, adecuadamente reglamentada, puede llegar a constituir una verdadera revolución en lo referente a la atención de la salud en nuestro país, al ampliarse la cobertura poblacional de la Seguridad Social y al sentarse las bases para que dicha atención sea de la mejor calidad científica, pero especialmente humana.

The energy conserving N5-methyltetrahydromethanopterin:coenzyme M methyltransferase complex from Methanobacterium thermoautotrophicum is composed of eight different subunits.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase (Mtr) from Methanobacterium thermoautotrophicum strain Marburg is a membrane-associated enzyme complex which catalyzes an energy-conserving, sodium-ion-translocating step in methanogenesis from H2 and CO2. We report here that the complex is composed of eight different subunits for which evidence was obtained at the protein, DNA and RNA levels: (a) SDS/PAGE of the purified complex revealed the presence of eight different polypeptides of apparent molecular masses of 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 (MtrD), 13 (MtrG), 12.5 (MtrB) and 12 kDa (MtrF). The N-terminal amino acid sequences of the 12-, 12.5- and 13-kDa polypeptides, which had previously not been accessible, were determined; (b) cloning and sequencing of the corresponding genes revealed the presence of the eight mtr genes organized in a 4.9-kbp gene cluster in the order mtrEDCBAFGH; (c) Northern-blot analysis revealed the presence of a 5-kbp transcript. DNA probes derived from the mtrE and mtrH genes hybridized to the transcript, indicating that the eight mtr genes are organized in a transcription unit. By primer extension, the 5' end of the mtrEDC-BAFGH mRNA was analyzed. The mtr operon was found to be located between the methyl-coenzyme M reductase I operon (mcr) and a downstream open reading frame predicted to encode a Na+/Ca2+, K+ exchanger.

The energetics and sodium-ion dependence of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase studied with cob(I)alamin as methyl acceptor and methylcob(III)alamin as methyl donor.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane-associated enzyme complex that uses a methyl-transfer reaction to drive an energy-conserving sodium-ion pump. Methyl transfer occurs in two steps, first from N5-methyltetrahydromethanopterin (CH3-H4MPT) to an enzyme-bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H-S-CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH3-H4MPT and cob(I)alamin (reaction a), and methyl-coenzyme M formation from methylcob(III)alamin and H-S-CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH3-H4MPT to H-S-CoM. The free energy changes (delta G0') associated with reactions a and b were both between -10 kJ/mol and -20 kJ/mol, consistent with a free energy change of approximately -30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium-ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme-bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H-S-CoM is a site of coupling with sodium-ion translocation.

N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanobacterium thermoautotrophicum. Catalytic mechanism and sodium ion dependence.

N5-Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane associated, corrinoid-containing enzyme complex which uses a methyl-transfer reaction to drive an energy-conserving sodium ion pump. The purified methyltransferase from Methanobacterium thermoautotrophicum (strain Marburg) exhibited a rhombic EPR signal indicative of a base-on cob(II)amide. In this form, the enzyme was almost completely inactive. Upon addition of Ti(III)citrate, which is a one-electron reductant known to reduce corrinoids to the cob(I)amide form, the EPR signal was completely quenched. In the reduced form, the enzyme was active. When the purified complex was incubated in the presence of both Ti(III) and N5-methyltetrahydromethanopterin (CH3-H4MPT), enzyme-bound Co-methyl-5'-hydroxybenzimidazolyl cob(III)amide was formed. Upon incubation of the methylated enzyme with either tetrahydromethanopterin or coenzyme M, the enzyme was demethylated with the concomitant formation of CH3-H4MPT and methylcoenzyme M, respectively. Enzyme demethylation, in contrast to enzyme methylation, was not dependent on the presence of Ti(III). Methyl transfer from the methylated enzyme to coenzyme M was essentially irreversible. These results are interpreted to that the purified enzyme complex is active only when the enzyme-bound corrinoid is in the reduced cob(I)amide form, and that methyl transfer from CH3-H4MPT to coenzyme M proceeds via nucleophilic attack of the cobalt(I) on the N5-methyl substituent of CH3-H4MPT, forming an enzyme-bound CH3-corrinoid as intermediate. Methyl-coenzyme M formation from CH3-H4MPT and coenzyme M, as catalyzed by the purified methyltransferase, was stimulated by sodium ions, half-maximal activity being obtained at approximately 50 microM Na+. We therefore infer that the methyltransferase, as isolated, is capable of vectorial sodium ion translocation.

Nucleotide sequence of a putative succinate dehydrogenase operon in Thermoplasma acidophilum.

28 amino acids from the N-terminal region of a putative terminal oxidase from the archaebacterium Thermoplasma acidophilum were determined by Edman degradation. On basis of this amino acid sequence a degenerated oligonucleotide was synthesized and used as a radioactive probe for Southern blot analysis of EcoRI digested genomic DNA. A 2.3 kb EcoRI fragment strongly hybridized to the probe and size selected genomic library from genomic DNA was constructed. Several clones scored positive by screening the library with the degenerated oligonucleotide, from which only one clone contained a EcoRI DNA fragment encoding the 28 amino acid sequence determined by protein sequencing. Sequence analysis revealed the presence of three genes in the typical arrangement of an operon. The first gene codes for a protein containing 11 cystein residues in an arrangement typical for Fe/S proteins. Protein sequence comparison revealed significant homologies to the fumarate reductase and succinate dehydrogenase of other bacteria. The two other genes encode small hydrophobic proteins probably serving as membrane anchor for the Thermoplasma acidophilum succinate dehydrogenase.

Purification and properties of N5-methyltetrahydromethanopterin:coenzyme M methyltransferase from Methanobacterium thermoautotrophicum.

N5-Methyltetrahydromethanopterin:coenzyme M meth-yltransferase is an integral membrane protein found in methanogenic archaea. It catalyzes an energy-conserving step in methane formation from CO2 and from acetate. The enzyme from Methanobacterium thermoautotrophicum (strain Marburg) has been purified 30-fold to apparent homogeneity. The purified enzyme had an apparent molecular mass of 670 kDa and was composed of seven different polypeptides of 34 kDa, 28 kDa, 24 kDa, 23 kDa, 21 kDa, 13 kDa, and 12 kDa. The N-terminal amino acid sequences of these polypeptides were determined. The native 670-kDa enzyme was found to contain 7.6 mol 5-hydroxybenzimidazolyl cobamide/mol, 37 mol non-heme iron/mol and 34 mol acid-labile sulfur/mol. Cobalt analyses after sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that the corrinoid was bound to the 23-kDa polypeptide. The apparent molecular masses of the polypeptides given above were determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis without boiling the samples prior to analysis. When the samples were boiled, as is usually done, the 23-kDa polypeptide changed its apparent molecular mass to 33 kDa and the 21-kDa, 24-kDa, and 28-kDa polypeptides formed aggregates. The specific activity (apparent Vmax) of the purified methyltransferase preparation was 11.6 mumol.min-1.mg protein-1. The apparent Km for N5-methyltetrahydromethanopterin was 260 microM and that for coenzyme M was 60 microM. The preparation was absolutely dependent on the presence of Ti(III) for activity. ATP enhanced the activity 1.5-2-fold.

Function of methylcobalamin: coenzyme M methyltransferase isoenzyme II in Methanosarcina barkeri.

Methanosarcina barkeri was recently shown to contain two cytoplasmic isoenzymes of methylcobalamin: coenzyme M methyltransferase (methyltransferase 2). Isoenzyme I predominated in methanol-grown cells and isoenzyme II in acetate-grown cells. It was therefore suggested that isoenzyme I functions in methanogenesis from methanol and isoenzyme II in methanogenesis from acetate. We report here that cells of M. barkeri grown on trimethylamine, H2/CO2, or acetate contain mainly isoenzyme II. These cells were found to have in common that they can catalyze the formation of methane from trimethylamine and H2, whereas only acetate-grown cells can mediate the formation of methane from acetate. Methanol-grown cells, which contained only low concentrations of isoenzyme II, were unable to mediate the formation of methane from both trimethylamine and acetate. These and other results suggest that isoenzyme II (i) is employed for methane formation from trimethylamine rather than from acetate, (ii) is constitutively expressed rather than trimethylamine-induced, and (iii) is repressed by methanol. The constitutive expression of isoenzyme II in acetate-grown M. barkeri can explain its presence in these cells. The N-terminal amino acid sequences of isoenzyme I and isoenzyme II were analyzed and found to be only 55% similar.