Metabolism of (+/-)-N-(n-propyl) amphetamine by Cunninghamella echinulata.

(+/-)-N-(n-propyl) amphetamine (I), a secondary amine, was readily metabolized by Cumminghamella echinulata. The products included known C- and N-oxygenated mammalian metabolites as well as N-acetylamphetamine and were identified by gas chromatography and mass spectrometry.     

Bioalkylation of dibenz[a,b]anthracene in rat liver cytosol

Previous studies by other investigators have established that L-region methyl derivatives of dibenz[a,h]anthracene (DBA) were more carcinogenic than the parent hydrocarbon. The bioalkylation of DBA was investigated by incubating the hydrocarbon with rat liver cytosol fortified with S-adenosyl-L-methionine (SAM) in 0.1 M phosphate buffer (pH 7.4) for 1 h at 37 degrees C in air. The reaction was stopped by the addition of cold acetone and the mixture extracted with ethyl acetate and washed with water. The organic phase was evaporated and the residue dissolved in methylene chloride for analysis by reverse phase high performance liquid chromatography (HPLC) and gas chromatography/mass spectroscopy GC/MS. Products were found that were indistinguishable from 7-methyl-DBA and 7,14-dimethyl-DBA, 7-hydroxymethyl-DBA, 7-hydroxymethyl-14-methyl-DBA, and 7,14-dihydroxymethyl-DBA. The results suggest that unsubstituted carcinogenic hydrocarbons are preprocarcinogens that react with SAM in liver cytosol preparations, to form alkyl substituted procarcinogens, which are more potent than the corresponding preprocarcinogens.     

Unwanted Horse Population in Illinois: Perceptions of Horse Owners,Non-Horse Owners,and Equine Industry Stakeholders

This paper presents the results of an investigation to determine perceptions, awareness, and knowledge of the unwanted horse population in Illinois from the viewpoint of horse owners, non-horse owners, and equine industry stakeholders. A questionnaire included items that pertained to knowledge of current legislation, equine background, current methods of controlling the unwanted horse population, and methods that respondents believe would reduce the unwanted horse population in Illinois. Results indicated that 58% of horse owners viewed horses as companion animals. Respondents perceived financial hardship to be the major reason why horses become unwanted. Current methods of managing unwanted horse populations were found to be ineffective. Reducing the costs of euthanasia and carcass disposal, allowing processing facilities to reopen in Illinois, and increasing the availability of gelding programs emerged as the most effective ways to manage the unwanted horse population. Results of this survey may lead to greater awareness of the unwanted horse population in Illinois. Furthermore, these results may lead to discussions about future legislation in the State designed to support and manage unwanted horses.     

Subfreezing volumetric behavior and stochastic modeling of intracellular ice formation in Drosophila melanogaster embryos

Cryomicroscopic observations were made of the volumetric behavior and kinetics of intracellular ice formation (IIF) in Drosophila melanogaster embryos in a modified cell culture medium (BD.20) or BD.20 + 2 M ethylene glycol. After rapid cooling to a given temperature, transient volumetric contraction of the embryos during the isothermal period was quantified by computerized video image analysis. Fitting these data to the numerical solution of the volume flux equation yielded estimates of the hydraulic permeability coefficient (Lp) for individual embryos at various subfreezing temperatures. Lp approximately followed an Arrhenius relation between -2 and -9 degrees C, with a value of 0.168 microns/(min-atm) extrapolated to 0 degrees C and an apparent activation energy delta E of 38.9 kcal/mol. IIF during an isothermal period occurred at random times whose characteristic temperature range and kinetics were affected by the presence of ethylene glycol. A stochastic process model developed to fit these data indicated the influence of both time-dependent and instantaneous components of IIF, presumed to be the result of seeding and heterogeneous nucleation, respectively. The presence of 2 M ethylene glycol depressed the characteristic temperature of instantaneous IIF by about 12 degrees C and reduced the rate constant for time-dependent IIF. Comparison with observed incidences of IIF yielded an estimate of the supercooling tolerance of 3 to 5 degrees C.     

Phosphorylation-regulated cleavage of the tumor suppressor PTEN by caspase-3: implications for the control of protein stability and PTEN-protein interactions

PTEN phosphatase is one of the most commonly targeted tumor suppressors in human cancers and a key regulator of cell growth and apoptosis. We have found that PTEN is cleaved by caspase-3 at several target sites, located in unstructured regions within the C terminus of the molecule. Cleavage of PTEN was increased upon TNFalpha-cell treatment and was negatively regulated by phosphorylation of the C-terminal tail of PTEN by the protein kinase CK2. The proteolytic PTEN fragments displayed reduced protein stability, and their capability to interact with the PTEN interacting scaffolding protein S-SCAM/MAGI-2 was lost. Interestingly, S-SCAM/MAGI-2 was also cleaved by caspase-3. Our findings suggest the existence of a regulatory mechanism of protein stability and PTEN-protein interactions during apoptosis, executed by caspase-3 in a PTEN phosphorylation-regulated manner.     

Changes in DNA conformation induced by gamma irradiation in the presence of copper

Gamma irradiation of DNA solutions containing copper causes changes in DNA conformation in oligonucleotides and in natural and synthetic DNAs. Diagnostic for these conformational changes is a ubiquitous 187-nm peak in the circular dichroism (CD) difference spectrum that has been predicted for a transformation from a right-handed to a left-handed helical DNA conformation. Changes in CD are correlated with changes in the UV spectrum. Reduction of DNA-bound Cu(II) to Cu(I) with ascorbic acid produces similar changes in CD spectra. These changes can be produced by the peroxy radical anion (O2*-) and the OH radical in the presence of copper. O2*- is approximately twice as efficient as *OH in initiating these changes in natural DNA. The changes in DNA conformation induced by ionizing radiation are remarkable in that they are dependent on the copper-ion concentration in a highly nonlinear manner at low copper concentrations and are not observed in the absence of copper ions. Possible implications of our results for radiobiological and oxidative damage in the cell nucleus are discussed.     

Evidence for an arginine residue at the substrate binding site of Escherichia coli adenylosuccinate synthetase as studied by chemical modification and site-directed mutagenesis

Chemical modification of adenylosuccinate synthetase from Escherichia coli with phenylglyoxal resulted in an inhibition of enzyme activity with a second-order rate constant of 13.6 M-1 min-1. The substrates, GTP or IMP, partially protected the enzyme against inactivation by the chemical modification. The other substrate, aspartate, had no such effect even at a high concentration. In the presence of both IMP and GTP during the modification, nearly complete protection of the enzyme against inactivation was observed. Stoichiometry studies with [7-14C]phenylglyoxal showed that only 1 reactive arginine residue was modified by the chemical reagent and that this arginine residue could be shielded by GTP and IMP. Sequence analysis of tryptic peptides indicated that Arg147 is the site of phenylglyoxal chemical modification. This arginine has been changed to leucine by site-directed mutagenesis. The mutant enzyme (R147L) showed increased Michaelis constants for IMP and GTP relative to the wild-type system, whereas the Km for aspartate exhibited a modest decrease as compared with the native enzyme. In addition, kcat of the R147L mutant decreased by a factor of 1.3 x 10(4). On the bases of these observations, it is suggested that Arg147 is critical for enzyme catalysis.