Studies on the biosynthesis of cholesterol—7 Rearrangement of methyl groups during enzymic cyclisation of squalene

The present state of knowledge of the biosynthesis of cholesterol is summarised. An experiment is described wherein a special application of the carbon isotope ¹³C permits a decision between possible modes of rearrangement during the enzymic cyclisation of squalene.     

Potential Mechanisms of Photodynamic Inactivation of Virus by Methylene BlueI. RNA–Protein Crosslinks and Other Oxidative Lesions in Qβ Bacteriophage

A spectrum of oxidative lesions was observed in a bacteriophage-based model system that is very sensitive to the photodynamic activity of selected dyes. When suspensions of the intact bacteriophage Qβ were exposed to methylene blue plus light (MB+L), inactivating events, or "hits" occurred that were oxygen-dependent and that were associated with the formation of several specific lesions: (1) carbonyl moieties on proteins, (2) 8-oxo-7,8-dihydroguanine (8-oxoGua), and (3) single-strand breaks (ssb) in the RNA genome and (4) RNA-protein crosslinks. Formation of carbonyl groups associated with protein in the Qβ phage preparation correlated positively with photoinactivation of the phage with increasing doses of either of the sensitizers MB or rose bengal. Strand breaks in the Qβ genomic RNA were observable at high MB concentrations but appeared not to be significant at the lower concentrations of MB, as full-length Qβ RNA was observable well beyond the 99% inactivation point in MB dosage. It was shown that the number of 8-oxoGua lesions were unlikely to be sufficient to account for the number of lethal events. Following exposure to MB+L, crosslink formation between Qβ RNA and protein was observed by virtue of the location of RNA at the interface of phenol-aqueous extractions of phage suspensions. A significant increase over background of RNA-protein complexes (including full-length Qβ RNA) was observed at the lowest concentration of MB tested (0.5 μ M ), which corresponded roughly to an average of 2 lethal hits per phage or approximately 13% survival compared to the zero MB control (100% survival). Due to its close correlation with Qβ inactivation and its expected lethality, RNA-protein crosslink formation may be important as an inactivating lesion in bacteriophage Qβ following MB+L exposure.     

Atmospheric Pressure Ionization Mass Spectrometry

Abstract

Atmospheric pressure ionization (API) mass spectrometry is a novel form of mass spectrometry in which the ionization process is carried out in a reaction chamber external to the mass analyzer region. The mass analyzer serves as a device to detect positive or negative ions present in the reaction chamber, which is maintained at atmospheric pressure.     

A method for the determination of d-kynurenine in biological tissues

d-Kynurenine (d-KYN), a metabolite of d-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of d-KYN in biological tissues, we developed a novel assay based on the conversion of d-KYN to KYNA by purified d-amino acid oxidase (d-AAO). Samples were incubated with d-AAO under optimal conditions for measuring d-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for d-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other d-amino acids, including d-serine and d-aspartate, were present in the incubation mixture at 50-fold higher concentrations than d-KYN. Using this new method, d-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with d-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for d-KYN measurement will be instrumental for evaluating whether d-KYN should be considered for a role in physiology and pathology.     

Energy deposition during electron-induced dissociation

We report studies of the internal energy deposited during activation of mass-selected ions through electron-ion collisions. Characteristic fragmentations of the molecular ion of limonene and W(CO) ^n/+ (n = 1–6) indicate that electron-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer proceeds via multiple collisions and that the average internal energy deposited during the activation process can be selected to be similar to that associated with electron-impact ionization. Control of the degree of ion excitation through selection of the electron energy, flux, and interaction time with the ions of interest is demonstrated, and advantages of this promising activation technique are discussed.     

Detection of diazepam in horse hair samples by mass spectrometric methods

A method for the detection of diazepam in horse hair samples by low resolution gas chromatography-mass spectrometry (GC-MS) was developed. Two other techniques, gas chromatography-high-resolution mass spectrometry (GC-HRMS) and high-performance liquid chromatography-atmospheric pressure chemical-ionisation mass spectrometry (HPLC-APCI-MS-MS) were applied on some selected samples. Sample preparation was performed according to a technique previously described for human hair, involving incubation with Sorensen buffer and solvent extraction. Hair samples from different sites such as coat on the neck, coat on the back, mane and tail were collected from two thoroughbreds which had received several dosages of diazepam corresponding to a total dose of 750 mg and 200 mg of diazepam respectively. In the first experiment, by low resolution GC-MS using single ion monitoring, diazepam was detected in the mane for at least 85 d after the last administration. In the second one, using the same method, diazepam was detected in the coat on the neck up to 25 d following the last administration. Low resolution GC-MS data were confirmed by the two other techniques. Furthermore, GC-HRMS even made possible the detection of diazepam up to 38 d after the administration of 200 mg of diazepam.     

A phase I, randomized study of combined IL-2 and therapeutic immunisation with antiretroviral therapy

Background  

Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection.