Metal-Catalyzed Hydration of 2-Pyridyloxirane

In the presence of CuII the hydration of 2-pyridyloxiran is accelerated 18,000-fold, and its reaction with Cl–, Br–, and MeO– becomes 100% regiospecific for β-attack

A small plasmid for recombination-based screening

We reported recently the construction of the 4.4-kb RoK-derived pMADI plasmid carrying supF [Stewart et al., Gene 106 (1991) 97-101] that does not share nt sequences with Co1E1 and therefore permits recombination-based screening of 2 libraries that contain Co1E1 sequences. Here we describe the construction of the 2.5-kb R6K-derived plasmid, pMAD3, that lacks the [pi]-encoding pir gene required for R6K replication. To supply [pi] [Inuzuka and Helinski, Proc. Natl. Acad. Sci. USA 75 (1978) 5381-5385] in trans we employed pPR1[Delta]22pir 116, referred to henceforth as pPR1 [McEachern et al., Proc. Natl. Acad. Sci. USA 86 (1989) 7942-7946; Dellis and Filutowicz J. Bacteriol. 173 (1991) 1279-1286]. Plasmid pMAD3 is small enough to be amplified readily by PCR [Saiki et al., Science 230 (1985) 1350-1354]. This permits the insertion of larger fragments and the retrieval of larger 2 inserts, as well as the use of a simplified PCR-based cloning protocol which utilizes annealing rather than ligation to create recombinants in pMAD3 [Nisson et al., PCR Methods and Applications 1(1991) 120-123].Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29693/1/0000025.pd

Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3001658Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO2). To explore possible parallels between the metabolism, covalent binding and toxicity of TA and thiobenzamide (TB) we exposed freshly isolated rat hepatocytes to [14C]-TASO or [13C2D3]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO2 is formed, whereas at least some of the TASO2 can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined

Do ceramic femoral heads reduce taper fretting corrosion in hip arthroplasty? A retrieval study.

BACKGROUND: Previous studies regarding modular head-neck taper corrosion were largely based on cobalt chrome (CoCr) alloy femoral heads. Less is known about head-neck taper corrosion with ceramic femoral heads. QUESTIONS/PURPOSES: We asked (1) whether ceramic heads resulted in less taper corrosion than CoCr heads; (2) what device and patient factors influence taper fretting corrosion; and (3) whether the mechanism of taper fretting corrosion in ceramic heads differs from that in CoCr heads. METHODS: One hundred femoral head-stem pairs were analyzed for evidence of fretting and corrosion using a visual scoring technique based on the severity and extent of fretting and corrosion damage observed at the taper. A matched cohort design was used in which 50 ceramic head-stem pairs were matched with 50 CoCr head-stem pairs based on implantation time, lateral offset, stem design, and flexural rigidity. RESULTS: Fretting and corrosion scores were lower for the stems in the ceramic head cohort (p=0.03). Stem alloy (p=0.004) and lower stem flexural rigidity (Spearman\u27s rho=-0.32, p=0.02) predicted stem fretting and corrosion damage in the ceramic head cohort but not in the metal head cohort. The mechanism of mechanically assisted crevice corrosion was similar in both cohorts although in the case of ceramic femoral heads, only one of the two surfaces (the male metal taper) engaged in the oxide abrasion and repassivation process. CONCLUSIONS: The results suggest that by using a ceramic femoral head, CoCr fretting and corrosion from the modular head-neck taper may be mitigated but not eliminated. CLINICAL RELEVANCE: The findings of this study support further study of the role of ceramic heads in potentially reducing femoral taper corrosion

Metabolism and Toxicity of Thioacetamide and Thioacetamide SOxide in Rat Hepatocytes

“This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3002719The hepatotoxicity of thioacetamide (TA) has been known since 1948. In rats, single doses cause centrilobular necrosis accompanied by increases in plasma transaminases and bilirubin. To elicit these effects TA requires oxidative bioactivation leading first to its S-oxide (TASO) and then to its chemically reactive S,S-dioxide (TASO2) which ultimately modifies amine-lipids and proteins. To generate a suite of liver proteins adducted by TA metabolites for proteomic analysis, and to reduce the need for both animals and labeled compounds, we treated isolated hepatocytes directly with TA. Surprisingly, TA was not toxic at concentrations up to 50 mM for 40 hr. On the other hand, TASO was highly toxic to isolated hepatocytes as indicated by LDH release, cellular morphology and vital staining with Hoechst 33342/propidium iodide. TASO toxicity was partially blocked by the CYP2E1 inhibitors diallyl sulfide and 4-methylpyrazole, and was strongly inhibited by TA. Significantly, we found that hepatocytes produce TA from TASO relatively efficiently by back-reduction. The covalent binding of [14C]-TASO is inhibited by unlabeled TA which acts as a “cold-trap” for [14C]-TA and prevents its re-oxidation to [14C]-TASO. This in turn increases the net consumption of [14C]-TASO despite the fact that its oxidation to TASO2 is inhibited. The potent inhibition of TASO oxidation by TA, coupled with the back-reduction of TASO and its futile redox cycling with TA may help explain phenomena previously interpreted as “saturation toxicokinetics” in the in vivo metabolism and toxicity of TA and TASO. The improved understanding of the metabolism and covalent binding of TA and TASO facilitates the use of hepatocytes to prepare protein adducts for target protein identification

Plasmids for recombination-based screening

To facilitate recombination-based screening, we constructed the ColE1-based plasmid, [pi]G4, that confers chloramphenicol resistance, contains a polylinker with multiple unique restriction enzyme recognition sequences, and contains the genetic marker, supF. To facilitate recombination-based screening followed by rapid DNA sequencing, we inserted the selectable marker, supF, into each of 20 high-copy-number (hcn) pUC-derived NoC plasmids that were designed for multiplex DNA sequencing. To facilitate recombination-based screening of common cDNA libraries that often contain ColE1 sequences, we constructed a supF-carrying plasmid whose replication was driven from an R6K replicon that does not share sequence homology with ColE1. Furthermore, we incorporated a useful polylinker and increased the copy number of this plasmid to create the 4.4-kb hcn plasmid, pMAD1. Thus, these plasmids allow: (1) background-free transformation of cells by a supF plasmid carrying an antibiotic-resistance marker; (2) simultaneous performance of the recombination-based assay and DNA sequencing; and (3) screening bacteriophage cDNA libraries that contain ColE1 sequences by recombination with a supF plasmid that is not homologous to ColE1 derivatives.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29122/1/0000161.pd

The murine situs inversus viscerum (iv) gene responsible for visceral asymmetry is linked tightly to the Igh-C cluster on chromosome 12

The iv gene controls left-right determination during murine organogenesis. To map this gene, we analyzed backcross progeny produced by mating (C57BL/6J x MEV/Ty)F1-iv/+ heterozygotes to C57BL/6J-iv homozygotes. Hybridization of a murine ecotropic virus probe and several homeotic box gene probes coupled with analysis of dominant visible markers enabled us to exclude the iv locus from much of the mouse genome. Spurred by a recent report that mapped the iv gene to mouse chromosome 12 which was not excluded by our previous work, we used the polymerase chain reaction on our larger cohort to determine that the iv gene is indeed linked tightly to the Igh-C locus on this chromosome: we observed 0/156 recombinants between the iv and Igh-C loci. Combining data from the two studies demonstrates that the murine iv gene is close (1/201 recombinants) to the Igh-C cluster on chromosome 12.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28508/1/0000305.pd