The relationship between physical observables and the potential energy surface in the He-HF system

This paper examines the effect of infinitesimal functional variations in a rigid rotor He-HF potential surface on several different types of observables: inelastic cross sections, rate constants, and rotational energy level populations. The dynamics and kinetic observables studied were found to be sensitive to a large number of Legendre components of the potential with the region of highest sensitivity dependent upon the energy or temperature as well as the states related by the individual observable. Sensitivity to the entire surface tends to show a large degree of structure due to competition among sensitivities to the individual potential components. Significant information loss has been observed in the transition from microscopic to macroscopic observables.     

Strategies for multi-site GLP studies

 A GLP study can be performed at more than one site. This is called a multi-site study. Although, the study is performed at different sites, it is still one study and must completely comply with the GLP principles. The fact that different activities are conducted at different sites implies that the planning, the organization and the communication are crucial for the success of the study. This means that all the staff involved should know their responsibilities and should have the knowledge and skills to realize all the phases of the study according to the GLP principles. To achieve a well managed multi-site study, several strategies for setting up such a study can be followed. This paper focuses on the responsibilities, communication, and collaboration of the personnel, which are involved in a multi-site study. Several case studies are highlighted, and we concluded that the basic communication triangle in a single-site GLP study between test facility management, study director, and the quality assurance unit should be extended to the communication among test facility and test site management, study director, principle investigator(s), and the quality assurance units at the test sites. Introduction Received: 14 August 2002 Accepted: 26 November 2002     

A new quantitative thermospray LC-MS method for nicotine and its metabolites in biological fluids

A rapid thermospray liquid chromatography-mass spectrometry (TSP LC-MS) method is described for the simultaneous determination of nicotine and 17 of its metabolites. Chemical ionization of nicotine and its metabolites separated by reversed-phase HPLC is achieved by postcolumn addition of ammonium acetate buffer with the filament of the ion source turned off. Quantification is accomplished by selectively monitoring the unique protonated molecular ion of each metabolite. Trideuterated cotinine serves as an internal standard. Linear responses for cotinine, demethylcotinine, and trans-3'-hydroxycotinine were observed over a concentration range of 20-8000 ng/mL, and 80-8000 ng/ml for nicotine and nicotine-1'-N-oxide. Of the 17 metabolites examined, only nicotine, cotinine, demethylcotinine, and trans-3'-hydroxycotinine were detected in smokers' urine.     

Novel products from attempted michael addition to 1-methyl-1,2,5,6-tetrahydropyridine-4-carbonitrile

The reaction of some phenols with the title compound, in the presence of sodium, gives 2-(2-hydroxyaryl_piperidine derivatives. Geometrical isomers have been separated, which differ in having an equatorial (A) or axial (B) cyano group on the piperidine chair (the methyl and aryl groups are equatorial in both forms). The x-ray crystallographic structures of an example of each of A and B are reported and the proton NMR spectra are assigned.     

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.     

EPR studies on the thiophenodithiazolyl radical, C4H2S3N

Selective chlorination of thiophene-2,3-dithiol with SO(2)Cl(2) generates the corresponding sulfenyl chloride, 2,3-C(4)H(2)S(SCl)(2). Subsequent condensation with Me(3)SiN(3) yields the thiophenodithiazolylium salt [C(4)H(2)S(3)N]Cl, [TDTA]Cl. The structure of the cation, TDTA+, was established by X-ray diffraction as both its AsF(6)(-) and HSO(4)(-) salts. Reduction of [TDTA]Cl with Ag powder yields the radical TDTA* which was characterised by X- and Q-band (9 and 34 GHz) EPR and ENDOR studies. The spin density distributions estimated from the EPR/ENDOR measurements were found to be in very good agreement with those determined by DFT (B3LYP/6-31G*) indicating that ca 10% of the spin density is delocalised onto the thiophene ring. Comparison of the spin density distributions in TDTA* and the isoelectronic trithiatriazapentalenyl radical C(2)S(3)N(3), TTTA*, indicates that replacement of N by C-H leads to a localisation of the spin density on the dithiazolyl ring.     

Acridinium ester chemiluminescence: pH dependent hydrolysis of reagents and flow injection analysis of hydrogen peroxide and glutamate

An automated analysis system is described for the measurement of hydrogen peroxide based on a chemiluminescence reaction with phenyl 10-methylacridinium-9-carboxylate (PMAC). A reversed FIA experimental arrangement is used to establish the operating conditions for the measurement of submicromolar levels of hydrogen peroxide. The carrier stream consists of hydrogen peroxide standards prepared in a pH 9.0, boric acid buffer and the flow rate for this carrier/sample stream is 4 ml/min. Twenty microliters of a 10 mM PMAC solution, prepared in a pH 3 phosphate buffer, are injected into the carrier/sample stream. Hydrogen peroxide mixes with the PMAC reagent in an incubation coil that is constructed by wrapping 107 cm of polyethylene tubing around a 1 cm o.d. plastic rod. The chemiluminescence reaction is then initiated by adding base just before the sample passes in front of a photomultiplier tube (PMT) detector. The calculated limit of detection (S/N = 3) for hydrogen peroxide is 0.25 M. In addition, the pH dependent hydrolysis of the PMAC reagent is characterized by an HPLC method which has been specifically developed for the separation and detection of the hydrolysis products of PMAC. Results indicate that a pH of 3.0 is required for long term stability of the PMAC reagent. Finally, this system has been successfully extended to the measurement of glutamate by coupling a bioreactor column of glutamate oxidase with the hydrogen peroxide detection scheme. A detection limit (S/N = 3) of 0.5 M has been established for glutamate with a throughput of 200 samples per hour.