Quantification of (+)-calanolide A, a novel and naturally occurring anti-HIV agent, by high-performance liquid chromatography in plasma from rat, dog and human

A HPLC method was validated for quantification of (+)-calanolide A (1), a novel anti-HIV agent, in rat, dog and human plasma. The synthetic intermediate (±)-12-oxocalanolide A (2) was found to be a suitable internal standard. Compounds were extracted from plasma using a solid-phase C₁₈ cartridge and quantified over the assay range of 12.5 to 800 ng/ml. The method was utilized to determine (+)-calanolide A pharmacokinetics in rats, dogs and humans. This is the first report of a validated HPLC assay for determination of (+)-calanolide A concentrations in rat and dog plasma as well as human plasma obtained from clinical trials. There was no evidence of in vivo epimerization of (+)-calanolide A to its inactive epimer (+)-calanolide B (3).     

Intracellular localisation and expression of mammalian CDC2 protein during myogenic differentiation

Myogenic differentiation involves withdrawal of myoblasts from the cell cycle and fusion to form multinucleate myotubes. To examine the role that cell cycle control genes may play in this process, we investigated the steady state levels of CDC2 protein and RNA during myogenesis of L6E9 rat myoblasts. Indirect immunofluorescence using a CDC2 affinity-purified antibody showed that this protein is localised exclusively in the cytoplasm with a higher concentration perinuclearly. Both protein and RNA levels were down-regulated to similar extents early in the differentiation process, as cells became quiescent. There was a further down-regulation of protein after fusion to form myotubes. Autonomous expression of CDC2 protein in L6E9 cells, after stable transfection with a metallothionein: CDC2 gene construct, failed to inhibit the differentiation process. This suggests that, although there is down-regulation in levels of CDC2 RNA and protein during myogenesis, this phenomenon per se does not play a primary role in controlling the differentiation process. If CDC2 is involved in control of differentiation, this must depend on post-translational modification of the protein.     

Improved RNA quality and TaqMan<Superscript>®</Superscript>Pre-amplification method (PreAmp) to enhance expression analysis from formalin fixed paraffin embedded (FFPE) materials

Background  

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an abundant source of clinical specimens; however their use is limited in applications involving analysis of gene expression due to RNA degradation and modification during fixation and processing. This study improved the quality of RNA extracted from FFPE by introducing a heating step into the selected extraction protocols. Further, it evaluated a novel pre-amplification system (PreAmp) designed to enhance expression analysis from tissue samples using assays with a range of amplicon size (62–164 bp).     

Images of mitochondrial UCP 1 in mouse thymocytes using confocal microscopy

The aim of this study was to demonstrate the constitutive expression of mitochondrial uncoupling protein 1 (UCP 1) in pure thymocytes using laser scanning confocal microscopic imagery. To that end we probed thymocytes from UCP 1 knock-out and wild-type mice. Mitochondrial location in thymocytes was determined using Mitotracker Red and the nucleus was labelled using Hoescht stain. We demonstrate that all cells investigated were thymocytes as determined by a monoclonal antibody specific for the thymocyte surface marker Thy 1 (CD90) pre-coupled to a fluorescent labelled (Alexa 448, green). Using a primary peptide antibody specific to UCP 1, and secondary fluorescently labelled (Alexa 647, magenta) antibody, we were able to demonstrate that UCP 1 is associated with mitochondria in thymocytes from UCP 1 wild-type mice but not thymocytes from UCP1-knock-out mice. These are the first images demonstrating the presence of UCP 1 in thymocyte mitochondria, in situ, and the first to clearly demonstrate UCP 1 expression in cells other than brown adipocytes. We conclude that mouse thymocytes contain UCP 1 in their mitochondria.     

Anti-HIV natural product (+)-calanolide A is active against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis

Naturally occurring anti-HIV-1 agent (+)-calanolide A was found to be active against all of the strains of Mycobacterium tuberculosis tested, including those resistant to the standard antitubercular drugs. Efficacy evaluations in macrophages revealed that (+)-calanolide A significantly inhibited intracellular replication of M. tuberculosis H37Rv at concentrations below the MIC observed in vitro. Preliminary mechanistic studies indicated that (+)-calanolide A rapidly inhibits RNA and DNA synthesis followed by an inhibition of protein synthesis. Compared with known inhibitors, this scenario is more similar to effects observed with rifampin, an inhibitor of RNA synthesis. Since (+)-calanolide A was active against a rifampin-resistant strain, it is believed that these two agents may involve different targets. (+)-Calanolide A and its related pyranocoumarins are the first class of compounds identified to possess antimycobacterial and antiretroviral activities, representing a new pharmacophore for anti-TB activity.     

Upregulation of Retinal Dehydrogenase 2 in Alternatively Activated Macrophages during Retinoid-dependent Type-2 Immunity to Helminth Infection in Mice

Although the vitamin A metabolite retinoic acid (RA) plays a critical role in immune function, RA synthesis during infection is poorly understood. Here, we show that retinal dehydrogenases (Raldh), required for the synthesis of RA, are induced during a retinoid-dependent type-2 immune response elicited by Schistosoma mansoni infection, but not during a retinoid-independent anti-viral immune response. Vitamin A deficient mice have a selective defect in T_H2 responses to S. mansoni, but retained normal LCMV specific T_H1 responses. A combination of in situ imaging, intra-vital imaging, and sort purification revealed that alternatively activated macrophages (AAMφ) express high levels of Raldh2 during S. mansoni infection. IL-4 induces Raldh2 expression in bone marrow-derived macrophages in vitro and peritoneal macrophages in vivo. Finally, in vivo derived AAMφ have an enhanced capacity to induce Foxp3 expression in CD4+ cells through an RA dependent mechanism, especially in combination with TGF-β. The regulation of Raldh enzymes during infection is pathogen specific and reflects differential requirements for RA during effector responses. Specifically, AAMφ are an inducible source of RA synthesis during helminth infections and T_H2 responses that may be important in regulating immune responses.     

Infection-Triggered Familial or Recurrent Cases of Acute Necrotizing Encephalopathy Caused by Mutations in a Component of the Nuclear Pore, RANBP2

Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy that can occur in otherwise healthy children after common viral infections such as influenza and parainfluenza. Most ANE is sporadic and nonrecurrent (isolated ANE). However, we identified a 7 Mb interval containing a susceptibility locus (ANE1) in a family segregating recurrent ANE as an incompletely penetrant, autosomal-dominant trait. We now report that all affected individuals and obligate carriers in this family are heterozygous for a missense mutation (c.1880C→T, p.Thr585Met) in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2). To determine whether this mutation is the susceptibility allele, we screened controls and other patients with ANE who are unrelated to the index family. Patients from 9 of 15 additional kindreds with familial or recurrent ANE had the identical mutation. It arose de novo in two families and independently in several other families. Two other patients with familial ANE had different RANBP2 missense mutations that altered conserved residues. None of the three RANBP2 missense mutations were found in 19 patients with isolated ANE or in unaffected controls. We conclude that missense mutations in RANBP2 are susceptibility alleles for familial and recurrent cases of ANE.