Publications

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.

This paper describes and compares 2 random-digit dialing (RDD) methods that have been used to select minority subjects for population-based research. These methods encompass the census-based method, which draws its primary sampling units from census tracts with a high proportion of minority persons, and the registry-based method, which derives its primary sampling units from a population-based cancer registry. Our study targeted Filipinos living in 10 Northern California counties, where they constitute 4% of the total population. Eligible participants (Filipina women, at least aged 20, who spoke 1 of 4 interview languages) were asked to complete a short telephone interview. Both the census and registry methods located Filipino households with comparable efficiency and with a higher yield than would be expected in a non-targeted population survey, such as the Mitofsky-Waksberg RDD method. No systematic pattern of responses was evident that would indicate that either method sampled women who were systematically less acculturated or less likely to use cancer screening tests. Although both methods offer substantial gains in efficiency, their utility is limited by generating samples that tend to over-represent high-density areas. The degree to which these methods are considered viable depends on further refinement to limit, or eliminate, their inherent selection biases without sacrificing their increased efficiency to locate minority populations.

Ferredoxin, Fd, is often deficient in metronidazole-resistant strains of Trichomonas vaginalis and is thought to be necessary for drug activation. To directly test whether Fd is essential for metronidazole susceptibility, gene replacement technology has been developed for T. vaginalis. The selectable marker gene neomycin phosphotransferase (NEO) flanked by approximately 2.6 and approximately 2.0 kBp of the Fd 5' and 3' flanking regions (pKO-FD-NEO) was introduced into cells on linear DNA and selected for NEO gene expression. Stable transformants were shown to contain the NEO gene in the Fd locus and to have completely lost the Fd gene. Northern and immunoblot analyses confirm the loss of Fd mRNA and protein in pKO-FD-NEO cells. Analyses of the activity of hydrogenosomal proteins in Fd KO cells show a fourfold increase in hydrogenase activity and a 95% decrease in pyruvate/ferredoxin oxidoreductase (PFO) activity. In contrast, PFO and hydrogenase mRNA levels are unchanged. Surprisingly, Fd KO cells are not resistant to metronidazole under aerobic or anaerobic conditions. These cells are capable of producing molecular hydrogen, albeit at 50% the level of the parental strain, demonstrating that the Fd gene product eliminated in KO cells is neither necessary for hydrogen production nor metronidazole activation. Together these data indicate the presence of unidentified Fds or flavodoxins capable of drug activation or an unidentified mechanism that does not require either PFO or Fd for metronidazole activation.