Reassignment of the guanine-binding mode of reduced mitomycin C

Mitomycin C (1) is a clinically used antitumor antibiotic that binds covalently to deoxyribonucleic acid under reductive or acidic catalysis. We have determined the structures of the adducts resulting from attack of reductively activated 1 on the dinucleoside phosphate d(GpC) to be N2-(2' beta, 7'-diaminomitosen-1'alpha-yl)-2'-deoxyguanosine (2) and its 1' beta-isomer (3). This represents a revision of the previously reported structures for these adducts in that the mitomycin residue is linked to the N2- rather than O6-position of 2'-deoxyguanosine. This revision is the result of applying to the mitomycin case a newly developed general method that leads to unambiguous assignment of the linkage position in complex alkylated guanosines. The method as described here takes advantage of the resolution enhancement gained by calculation of the second derivatives of absorbance Fourier transform infrared spectra. In addition, we present 1H NMR data that corroborate the assigned structures of 2 and 3 and that should serve as a useful reference for future investigations into the binding of mitomycin C to DNA. The convenient synthesis of adducts 2 and 3 from deoxyguanosine and mitomycin C reported here should facilitate such investigations as well. Furthermore, we demonstrate a useful acetylation procedure for adducts and metabolites of mitomycin C that furnishes spectroscopically superior chemical derivatives (e.g., triacetates 4 and 5, derived from acetylation of adducts 2 and 3).     

Differences in evolutionary pressure acting within highly conserved ortholog groups

Background  

In highly conserved widely distributed ortholog groups, the main evolutionary force is assumed to be purifying selection that enforces sequence conservation, with most divergence occurring by accumulation of neutral substitutions. Using a set of ortholog groups from prokaryotes, with a single representative in each studied organism, we asked the question if this evolutionary pressure is acting similarly on different subgroups of orthologs defined as major lineages (e.g. Proteobacteria or Firmicutes).     

Utilization of cholestyramine resin as a preventive treatment for antibiotic (clindamycin) induced enterotoxaemia in the rabbit.

Cholestyramine, an ion exchange resin shown to bind bacterial toxins, was utilized to treat rabbits with antibiotic induced enterotoxaemia. Three groups of 6 rabbits were administered 30 mg/kg clindamycin phosphate intravenously on day 1. One group was untreated; 2 groups were treated daily by gavage with 2 g cholestyramine in 20 ml water until day 21, starting on either day 1 or 3. Daily body weights, faecal output, faecal occult blood, food and water consumption, and body temperatures were determined. Four of 6 rabbits in the untreated group either died or were moribund and euthanased. There were no deaths in either treatment groups. Dramatic decreases in food consumption (86%), water consumption (62%), and faecal output (89%) were noted within 3 days after clindamycin administration in all groups. These parameters remained depressed throughout the study. There was no clear trend in body weight changes, body temperature, or faecal occult blood test results. Cholestyramine was effective in eliminating mortality associated with the intravenous administration of clindamycin and is recommended to prevent the development of enterotoxaemia when pyrogen testing or administering antibiotics known to induce the syndrome in rabbits.     

Line Transect Sampling of Primates: Can Animal-to-Observer Distance Methods Work?

Line transect sampling is widely used for estimating abundance of primate populations. Researchers commonly use animal-to-observer distances (AODs) in analysis, in preference to perpendicular distances from the line, which is in marked contrast with standard practice for other applications of line transect sampling. We formalize the mathematical shortcomings of approaches based on AODs, and show that they are likely to give strongly biased estimates of density. We review papers that claim good performance for the method, and explore this performance through simulations. These confirm strong bias in estimates of density using AODs. We conclude that AOD methods are conceptually flawed, and that they cannot in general provide valid estimates of density.     

Cathepsin B but not cathepsins L or S contributes to the pathogenesis of Unverricht‐Lundborg progressive myoclonus epilepsy (EPM1)

The inherited epilepsy Unverricht‐Lundborg disease (EPM1) is caused by loss‐of‐function mutations in the cysteine protease inhibitor, cystatin B. Because cystatin B inhibits a class of lysosomal cysteine proteases called cathepsins, we hypothesized that increased proteolysis by one or more of these cathepsins is likely to be responsible for the seizure, ataxia, and neuronal apoptosis phenotypes characteristic of EPM1. To test this hypothesis and to identify which cysteine cathepsins contribute to EPM1, we have genetically removed three candidate cathepsins from cystatin B‐deficient mice and tested for rescue of their EPM1 phenotypes. Whereas removal of cathepsins L or S from cystatin B‐deficient mice did not ameliorate any aspect of the EPM1 phenotype, removal of cathepsin B resulted in a 36–89% reduction in the amount of cerebellar granule cell apoptosis depending on mouse age. The incidence of an incompletely penetrant eye phenotype was also reduced upon removal of cathepsin B. Because the apoptosis and eye phenotypes were not abolished completely and the ataxia and seizure phenotypes experienced by cystatin B‐deficient animals were not diminished, this suggests that another molecule besides cathepsin B is also responsible for the pathogenesis, or that another molecule can partially compensate for cathepsin B function. These findings establish cathepsin B as a contributor to the apoptotic phenotype of cystatin B‐deficient mice and humans with EPM1. They also suggest that the identification of cathepsin B substrates may further reveal the molecular basis for EPM1. © 2003 Wiley Periodicals, Inc. J Neurobiol 56: 315–327, 2003