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1.  Absorption and Metabolism of cis-9,trans-11-CLA and of Its Oxidation Product 9,11-Furan Fatty Acid by Caco-2 Cells 
Lipids  2012;47(4):435-442.
Furan fatty acids (furan-FA) can be formed by auto-oxidation of conjugated linoleic acids (CLA) and may therefore be ingested when CLA-containing foodstuff is consumed. Due to the presence of a furan ring structure, furan-FA may have toxic properties, however, these substances are toxicologically not well characterized so far. Here we show that 9,11-furan-FA, the oxidation product of the major CLA isomer cis-9,trans-11-CLA (c9,t11-CLA), is not toxic to human intestinal Caco-2 cells up to a level of 100 μM. Oil-Red-O staining indicated that 9,11-furan-FA as well as c9,t11-CLA and linoleic acid are taken up by the cells and stored in the form of triglycerides in lipid droplets. Chemical analysis of total cellular lipids revealed that 9,11-furan-FA is partially elongated probably by the enzymatic activity of cellular fatty acid elongases whereas c9,t11-CLA is partially converted to other isomers such as c9,c11-CLA or t9,t11-CLA. In the case of 9,11-furan-FA, there is no indication for any modification or activation of the furan ring system. From these results, we conclude that 9,11-furan-FA has no properties of toxicological relevance at least for Caco-2 cells which serve as a model for enterocytes of the human small intestine.
doi:10.1007/s11745-012-3653-6
PMCID: PMC3311842  PMID: 22249938
Furan fatty acid; Conjugated linoleic acid; CLA; Caco-2
2.  Involvement of hyp Gene Products in Maturation of the H2-Sensing [NiFe] Hydrogenase of Ralstonia eutropha 
Journal of Bacteriology  2001;183(24):7087-7093.
The biosynthesis of [NiFe] hydrogenases is a complex process that requires the function of the Hyp proteins HypA, HypB, HypC, HypD, HypE, HypF, and HypX for assembly of the H2-activating [NiFe] site. In this study we examined the maturation of the regulatory hydrogenase (RH) of Ralstonia eutropha. The RH is a H2-sensing [NiFe] hydrogenase and is required as a constituent of a signal transduction chain for the expression of two energy-linked [NiFe] hydrogenases. Here we demonstrate that the RH regulatory activity was barely affected by mutations in hypA, hypB, hypC, and hypX and was not substantially diminished in hypD- and hypE-deficient strains. The lack of HypF, however, resulted in a 90% decrease of the RH regulatory activity. Fourier transform infrared spectroscopy and the incorporation of 63Ni into the RH from overproducing cells revealed that the assembly of the [NiFe] active site is dependent on all Hyp functions, with the exception of HypX. We conclude that the entire Hyp apparatus (HypA, HypB, HypC, HypD, HypE, and HypF) is involved in an efficient incorporation of the [NiFe] center into the RH.
doi:10.1128/JB.183.24.7087-7093.2001
PMCID: PMC95556  PMID: 11717266
3.  The H2 Sensor of Ralstonia eutropha Is a Member of the Subclass of Regulatory [NiFe] Hydrogenases 
Journal of Bacteriology  2000;182(10):2716-2724.
Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H2, which is available in low amounts in aerobic environments. H2 sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H2 sensor of R. eutropha is a cytoplasmic protein. Although capable of H2 oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for R. eutropha provided the basis for identifying the H2 sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231–235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H2 sensor is nickel dependent. The results suggest that H2 sensing requires an active [NiFe] hydrogenase, leaving the question open whether only H2 binding or subsequent H2 oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H2-sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H2 sensors represents a novel subclass of [NiFe] hydrogenases denoted as the “regulatory hydrogenases.”
PMCID: PMC101976  PMID: 10781538
4.  Positive Transcriptional Feedback Controls Hydrogenase Expression in Alcaligenes eutrophus H16 
Journal of Bacteriology  1999;181(18):5684-5692.
The protein HoxA is the central regulator of the Alcaligenes eutrophus H16 hox regulon, which encodes two hydrogenases, a nickel permease and several accessory proteins required for hydrogenase biosynthesis. Expression of the regulatory gene hoxA was analyzed. Screening of an 8-kb region upstream of hoxA with a promoter probe vector localized four promoter activities. One of these was found in the region immediately 5′ of hoxA; the others were correlated with the nickel metabolism genes hypA1, hypB1, and hypX. All four activities were independent of HoxA and of the minor transcription factor ς54. Translational fusions revealed that hoxA is expressed constitutively at low levels. In contrast to these findings, immunoblotting studies revealed a clear fluctuation in the HoxA pool in response to conditions which induce the hox regulon. Quantitative transcript assays indicated elevated levels of hyp mRNA under hydrogenase-derepressing conditions. Using interposon mutagenesis, we showed that the activity of a remote promoter is required for hydrogenase expression and autotrophic growth. Site-directed mutagenesis revealed that PMBH, which directs transcription of the structural genes of the membrane-bound hydrogenase, contributes to the expression of hoxA under hydrogenase-derepressing conditions. Thus, expression of the hox regulon is governed by a positive feedback loop mediating amplification of the regulator HoxA. These results imply the existence of an unusually large (ca. 17,000-nucleotide) transcript.
PMCID: PMC94088  PMID: 10482509

Results 1-4 (4)