A cell line with decreased sensitivity to the methyl mercury-induced stimulation of alpha-amanitin sensitive RNA synthesis in isolated nuclei

1. In nuclei isolated from cells of the B50 rat neuroblastoma line the stimulatory effect of methyl mercury on alpha-amanitin-sensitive RNA synthesis is very much reduced compared to the stimulatory effect in HeLa nuclei (see: Frenkel G. D. and Randles K. (1982) Specific stimulation of alpha-amanitin-sensitive RNA synthesis in isolated HeLa nuclei by methyl mercury. J. biol. Chem. 257, 6275-6279). 2. The stimulatory effect of another mercury compound, p-hydroxymercuribenzoate, was also much less pronounced in the B50 nuclei. 3. Similar results were obtained with nuclei isolated from B50 cells which had been induced to differentiate by exposure to dibutaryl cyclic AMP. 4. Nuclei isolated from cells of another rat neuroblastoma line (B35), and nuclei from cells of a human neuroblastoma line both exhibited levels of stimulation similar to that of HeLa nuclei. 5. The B50 and HeLa cells were also compared as to their sensitivity to other effects of methyl mercury.     

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.     

Effect of vitamin B-12 deficiency on the hepatic tissue concentration of acyl carnitines.

Concentrations of carnitine, acetyl carnitine, propionyl carnitine, and long chain acyl carnitines have been measured in hepatic tissue of normal and vitamin B-12 deficient rats using radiolabelled butyrobetaine to label carnitine pools. Increased levels of propionyl carnitine were seen in the livers of vitamin B-12 deprived animals when compared to those from normal animals. Methylmalonyl carnitine was not detected in the B-12 deprived animals. Free carnitine levels were no different in the livers from the B-12 deprived animals than from the normal control animals.