Antimitotic activity of dehydroretronecine, a pyrrolizidine alkaloid metabolite, and some analogous compounds, in a rat liver parenchymal cell line

The actions of 13 pyrrolic alcohols with similar chemical properties have been tested on cultured liver cells. Two, dehydroretronecine and dehydrosupinidine, were putative metabolites of hepatotoxic pyrrolizidine alkaloids; the remainder were synthetic. All were either mono- or bi-functional alkylating agents.

Groups of cells were exposed to the compounds and were later stimulated to divide by changing the medium, then fixed, stained, and the proportions of cells in mitosis counted and compared with those in similarly treated control cells.

Eleven compounds partially or completely inhibited cell division at concentrations of 10⁻⁴ M or less. Bifunctional compounds, including dehydroretronecine and 2,3-bis-hydroxymethyl-1-methylpyrrole, had the highest antimitotic activity coupled with lowest cytotoxicity. The least chemically reactive compound, 3-hydroxymethyl-1-methylpyrrole, was neither antimitotic nor cytotoxic, whereas the monofunctional alkylating agents with highest reactivity, such as 3-hydroxymethyl-1,2-dimethylpyrrole, were the most toxic to the cells.

The mitotic block occurred at a post-synthetic stage of the cell cycle, and affected cells could grow to a giant size.     

Review of massively parallel DNA sequencing technologies

Since the development of technologies that can determine the base-pair sequence of DNA, the ability to sequence genes has contributed much to science and medicine. However, it has remained a relatively costly and laborious process, hindering its use as a routine biomedical tool. Recent times are seeing rapid developments in this field, both in the availability of novel sequencing platforms, as well as supporting technologies involved in processes such as targeting and data analysis. This is leading to significant reductions in the cost of sequencing a human genome and the potential for its use as a routine biomedical tool. This review is a snapshot of this rapidly moving field examining the current state of the art, forthcoming developments and some of the issues still to be resolved prior to the use of new sequencing technologies in routine clinical diagnosis.     

Mitochondrial Hyperfusion during Oxidative Stress Is Coupled to a Dysregulation in Calcium Handling within a C2C12 Cell Model

Atrial Fibrillation is the most common sustained cardiac arrhythmia worldwide harming millions of people every year. Atrial Fibrillation (AF) abruptly induces rapid conduction between atrial myocytes which is associated with oxidative stress and abnormal calcium handling. Unfortunately this new equilibrium promotes perpetuation of the arrhythmia. Recently, in addition to being the major source of oxidative stress within cells, mitochondria have been observed to fuse, forming mitochondrial networks and attach to intracellular calcium stores in response to cellular stress. We sought to identify a potential role for rapid stimulation, oxidative stress and mitochondrial hyperfusion in acute changes to myocyte calcium handling. In addition we hoped to link altered calcium handling to increased sarcoplasmic reticulum (SR)-mitochondrial contacts, the so-called mitochondrial associated membrane (MAM). We selected the C2C12 murine myotube model as it has previously been successfully used to investigate mitochondrial dynamics and has a myofibrillar system similar to atrial myocytes. We observed that rapid stimulation of C2C12 cells resulted in mitochondrial hyperfusion and increased mitochondrial colocalisation with calcium stores. Inhibition of mitochondrial fission by transfection of mutant DRP1K38E resulted in similar effects on mitochondrial fusion, SR colocalisation and altered calcium handling. Interestingly the effects of ‘forced fusion’ were reversed by co-incubation with the reducing agent N-Acetyl cysteine (NAC). Subsequently we demonstrated that oxidative stress resulted in similar reversible increases in mitochondrial fusion, SR-colocalisation and altered calcium handling. Finally, we believe we have identified that myocyte calcium handling is reliant on baseline levels of reactive oxygen species as co-incubation with NAC both reversed and retarded myocyte response to caffeine induced calcium release and re-uptake. Based on these results we conclude that the coordinate regulation of mitochondrial fusion and MAM contacts may form a point source for stress-induced arrhythmogenesis. We believe that the MAM merits further investigation as a therapeutic target in AF-induced remodelling.     

Metabolism and toxicity of synthetic analogues of macrocyclic diester pyrrolizidine alkaloids

Seven macrocyclic diesters analogous to hepatotoxic pyrrolizidine alkaloids have been tested in male weanling Wistar rats. The compounds were the succinate (VII), 2,3-dimethylsuccinate (VIII), phthalate (IX), glutarate (X), 2,4-dimethylglutarate (XI), 3,3-dimethylglutarate (XII) and 3,3-pentamethyleneglutarate (XIII) of the synthetic amino dialcohol, synthanecine A. Single doses of these compounds were given i.p. to rats, and liver levels of pyrrolic metabolites were measured 2 h later. For these experiments both normal rats and rats pretreated with the esterase inhibitor tri-orthocresylphosphate (TOCP) were used. In normal rats, low levels of pyrrolic metabolites were formed from compounds VII, IX, X and XI, but these levels were greatly enhanced in rats with inhibited esterase activity. Much higher pyrrole levels were formed from compounds VIII, XII and XIII in normal rats, and esterase inhibition had relatively little effect on their metabolic conversion to pyrroles. This indicated that the last mentioned compounds were relatively resistant to enzymic hydrolysis, whereas VII, IX, X and XI were easily hydrolysed in normal rats, providing an alternative metabolic path which limited their conversion to pyrrolic metabolites. Comparison of results obtained using the 2,4-dimethylglutarate (XI), the 3,3-dimethylglutarate (XII) and the 3,3,-pentamethyleneglutarate (XIII) showed that 3,3-disubstitution but not 2,4-disubstitution in the glutaric acid moiety conferred high resistance to esterase attack. Toxicity tests using four of the compounds confirmed that acute hepatotoxicity was dose related, and associated with the formation of pyrrolic metabolites in the liver. The 3,3-dimethylglutarate (XII) was highly toxic both in normal and in TOCP treated rats, doses of 25-30 mg/kg causing moderate to severe centrilobular necrosis of the liver. In contrast the toxicity of the unsubstituted succinate (VII), glutarate (X) and 2,4-dimethylglutarate (XI) was very low in normal rats but high in rats with inhibited esterase activity. Thus, the glutarate (X) was non-toxic at 200 mg/kg in normal rats, but in TOCP treated rats, in which pyrrolic metabolite formation was enhanced by a factor of 17.5, a 50 mg/kg dose of this compound was severely hepatotoxic. Kidney damage, which was generally limited to the presence of isolated necrotic cells, sometimes accompanied the liver damage caused by these compounds, but acute toxic effects were not observed in any other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

RecFOR Is Not Required for Pneumococcal Transformation but Together with XerS for Resolution of Chromosome Dimers Frequently Formed in the Process

Homologous recombination (HR) is required for both genome maintenance and generation of diversity in eukaryotes and prokaryotes. This process initiates from single-stranded (ss) DNA and is driven by a universal recombinase, which promotes strand exchange between homologous sequences. The bacterial recombinase, RecA, is loaded onto ssDNA by recombinase loaders, RecBCD and RecFOR for genome maintenance. DprA was recently proposed as a third loader dedicated to genetic transformation. Here we assessed the role of RecFOR in transformation of the human pathogen Streptococcus pneumoniae. We firstly established that RecFOR proteins are not required for plasmid transformation, strongly suggesting that DprA ensures annealing of plasmid single-strands internalized in the process. We then observed no reduction in chromosomal transformation using a PCR fragment as donor, contrasting with the 10,000-fold drop in dprA ^- cells and demonstrating that RecFOR play no role in transformation. However, a ∼1.45-fold drop in transformation was observed with total chromosomal DNA in recFOR mutants. To account for this limited deficit, we hypothesized that transformation with chromosomal DNA stimulated unexpectedly high frequency (>30% of cells) formation of chromosome dimers as an intermediate in the generation of tandem duplications, and that RecFOR were crucial for dimer resolution. We validated this hypothesis, showing that the site-specific recombinase XerS was also crucial for dimer resolution. An even higher frequency of dimer formation (>80% of cells) was promoted by interspecies transformation with Streptococcus mitis chromosomal DNA, which contains numerous inversions compared to pneumococcal chromosome, each potentially promoting dimerization. In the absence of RecFOR and XerS, dimers persist, as confirmed by DAPI staining, and can limit the efficiency of transformation, since resulting in loss of transformant chromosome. These findings strengthen the view that different HR machineries exist for genome maintenance and transformation in pneumococci. These observations presumably apply to most naturally transformable species.     

Simultaneous chiral separation of methylamphetamine and common precursors using gas chromatography/mass spectrometry

Methylamphetamine, ephedrine, and pseudoephedrine were derivatized using trifluoroacetic anhydride and enantiomers of each were analyzed using gas chromatography coupled to mass spectrometry (GC/MS) fitted with a γ‐cyclodextrin (Chiraldex™ G‐PN) chiral column. A temperature‐programmed method was developed and optimized and the results compared with those obtained using a previously published isothermal GC method applied to GC/MS analysis. Trifluoroacetylated 3‐(trifluoromethyl)phenethylamine hydrochloride was used as an internal standard, and mass fragmentation patterns are proposed for all derivatives analyzed. Qualitative validation of the optimized chromatographic conditions was completed in accordance with the guidelines published by the United Nations Office on Drugs and Crime (UNODC). Under conditions of repeatability and reproducibility, the method gave relative retention times with a relative standard deviation of less than 0.02% for all six analytes of interest. This surpasses the UNODC's acceptance criteria of 2% for validation of qualitative precision. Ephedrine and pseudoephedrine are common precursors in the clandestine manufacture of methylamphetamine. Seizures of illicit methylamphetamine therefore often contain mixtures of these optically active compounds. The simultaneous enantioseparation of these compounds to produce a profile would provide valuable information to law enforcement agencies regarding the provenance of a methylamphetamine seizure. Chirality, 2011. © 2011 Wiley‐Liss, Inc.